Enzyme engineering to alter the functional repertoire of cannabinoid synthases

ABSTRACT

Described herein are variant, novel cannabinoid synthases, nucleic acids encoding same, and various uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/661,524 filed Apr. 23, 2018 which is incorporated herein byreference.

BACKGROUND Field of the Invention

The present disclosure relates to enzyme engineering to produce variantcannabinoid synthases.

Related Art

Cannabinoids are terpenophenolic secondary metabolites, produced byCannabis sativa (C. sativa) plants in the sessile and stalked trichomes.The steps involved in the biosynthesis of the different cannabinoidsfrom the common precursor have been largely elucidated. According tothis widely accepted pathway, cannabigerolic acid (CBGA) is the firstcannabinoid produced in the C. sativa cannabinoid biosynthesis pathway,formed through the condensation of a phenolic moiety (e.g. olivetolic ordivarinic acid) with the terpenoid component, geranyl pyrophosphate(GPP). CBGA and its alkyl homologs are considered as the commonprecursors of all the main cannabinoids produced through an enzymeactivity by the plant: i.e. the alkyl homologs of delta9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) andcannabichromenic acid (CBCA). All the CBGA alkyl-homologs can be used assubstrate and transformed by plant extracts containing the differentcannabinoid synthases in vitro, although the efficiency of conversionwas known to be different for each homolog. The different synthasescatalyzing the oxidocyclization of CBGA into THCA, CBDA or CBCA (andtheir alkyl homologs), have been characterized in recent years.THCA-synthase (THCAS) and CBDA-synthase (CBDAS) share many similaritiesin their biochemical properties, such as their protein mass (they aremonomeric proteins, approximately 74 kDa absent post-translationalmodification), pI, Vmax and Km for CBGA (while cannabichromenic acidsynthase has less information in public databases). They are bothsoluble enzymes with ˜84% sequence identity at the amino acid level(comparison based on GenBank accession numbers E55107 and E33090). Bothhave a 28-amino-acids putative signal peptide that is removed duringmaturation in the plant, and a FAD-binding domain. The tertiarystructure of THCAS was recently resolved and amino-acid positionsputatively involved in flavin adenine dinucleotide (FAD) and substratebinding were identified by X-ray crystallography to a 2.75 Å resolutionand also by mutational analysis. Natural sequence variants of THCAS andCBDAS have been isolated from different C. sativa strains and were shownto have different specificity and/or ability to convert the precursorCBGA into CBDA and THCA (and other products). The amount of nucleotide(and amino acid) diversity was found to be higher within the CBDASsequence family than in the THCAS family. It is therefore believed thatCBDAS is the ancestral type of these synthases.

Although natural products continue to provide about half of all newchemical entities approved as drugs by the US Food and DrugAdministration, industry efforts for drug discovery during the latterpart of the 20th century shifted away from exploring natural productsand instead shifted towards screening synthetic libraries. This paradigmshift reflected the complexity of small, natural libraries against thesimplicity of large, combinatorial synthetic libraries and wasrationalized to accommodate the enormous capacity of industrialhigh-throughput screening programs. The putative promised plethora ofnew drugs from combinatorial chemical libraries, however, did notmaterialize during this time period, while natural products continued toprove an important source of drug targets. Natural products likesphytocannabinoids that can accent the endocannabinoids pathway in humanscould serve as new drugs, represent a chemical space that thosecombinatorial compound libraries have not been able to tap into; andnatural phytocannabinoids have already shown powerful biology byactivating cell receptors throughout the human body and are capable ofpassing the blood-brain.

Plant derived cannabinoids (phytocannabinoids) from C. sativa consist ofa large family of over 100 natural molecules that happen to interactwith normal cell receptors throughout the human body (endocannabinoidreceptors) and are capable of passing the blood-brain barrier tointeract with receptors in the brain. Only a few of thesephytocannabinoids are psychoactive (such as Δ9-THC, a.k.a. THC), whilemany phytocannabinoids are not psychoactive. The cannabinoidΔ9-tetrahydrocannabinol (Δ9-THC) is made in large quantities in C.sativa and has been well studied for its interaction with receptors inthe brain. Medical research on non-psychoactive phytocannabinoidssuggest that these molecules have medicinal properties however many ofthese (aside from CBD) are produced by the plant at very low levels(i.e. “non-abundant cannabinoids”). These desirable non-abundantcannabinoids are not easily separated into pure forms after extractionfrom the plant (C. sativa), and so obtaining these non-abundantcannabinoids in industrial pure forms is often cost prohibitive. Thedifferent native cannabinoid synthases to C. sativa that catalyze theoxidocyclization of CBGA (and their alkyl homologs), into THCA, CBDA orCBCA have been characterized in recent years have proven to be difficultto express exogenously.

Because wild type cannabinoid synthases have various limitations, thereis a need for novel cannabinoid synthases, systems, and methods of usethat overcomes these limitations.

SUMMARY

Described herein, in certain embodiments, are variant cannabinoidsynthases or active fragments thereof comprising a non-naturallyoccurring amino acid sequence relative to a wild-type cannabinoidsynthase or an active fragment thereof which acts on a substrate toproduce an altered amount of a cannabinoid relative to an amount of thecannabinoid produced by the wild-type cannabinoid synthase or activefragment thereof In some embodiments, the variant cannabinoid synthaseis a variant cannabidiolic acid (CBDA) synthase comprising anon-naturally occurring amino acid sequence relative to a wild type CBDAsynthase of SEQ ID NO: 1043. In some embodiments, the variantcannabinoid synthase is a variant cannabidiolic acid (CBDA) synthasecomprising a non-naturally occurring amino acid sequence relative to awild type consensus CBDA synthase of SEQ ID NO: 1046. In someembodiments, the variant cannabinoid synthase is atetrahydrocannabinolic acid (THCA) synthase comprising a non-naturallyoccurring amino acid sequence relative to a wild type THCA synthase ofSEQ ID NO: 1044. In some embodiments, the variant cannabinoid synthaseis a tetrahydrocannabinolic acid (THCA) synthase comprising anon-naturally occurring amino acid sequence relative to a wild typeconsensus THCA synthase of SEQ ID NO: 1047. In some embodiments, thevariant cannabinoid synthase is a cannabichromenic acid (CBCA) synthasecomprising a non-naturally occurring amino acid sequence relative to awild type CBCA synthase of SEQ ID NO: 1045. In some embodiments, thevariant cannabinoid synthase is a cannabichromenic acid (CBCA) synthasecomprising a non-naturally occurring amino acid sequence relative to awild type consensus CBCA synthase of SEQ ID NO: 1048.

In some embodiments, the cannabinoid is selected from the groupconsisting of: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol(THC), tetrahydrocannabinvarin (THCV), cannabidiolic acid (CBDA),cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC),cannabichromevarin (CBCV), cannabichromenic acid (CBCA), and acombination thereof. In some embodiments, the substrate is a naturallyoccurring substrate. In some embodiments, the naturally occurringsubstrate is selected from the group consisting of cannabigerol (CBG),cannabigerolic acid (CBGA), cannbigerovarinic acid (GBGVA), and anyhomolog thereof. In some embodiments, the substrate is a non-naturallyoccurring substrate. In some embodiments, the non-naturally occurringsubstrate can comprise: a non-naturally occurring tail variant, a prenyldonor, or a combination thereof.

In some embodiments, the altered amount of the cannabinoid produces achange in a proportion of a first cannabinoid to a second cannabinoid.In some embodiments, the variant cannabinoid synthase is a variant CBDAsynthase. In some embodiments, the first cannabinoid is CBDA and thesecond cannabinoid is THCA. In some embodiments, a proportion ofCBDA:THCA produced by a wild type CBDA synthase is about 95:5. In someembodiments, the non-naturally occurring amino acid sequence comprisesan amino acid mutation at position 445 relative to a wild-type consensusCBDA synthase amino acid sequence set forth in SEQ ID NO: 1046. In someembodiments, the amino acid mutation at position 445 is selected fromthe group consisting of: I445M and I445L. In some embodiments, thealtered amount of the cannabinoid is an increase or a decrease in ayield of the cannabinoid. In some embodiments, the yield is a nMol ofthe cannabinoid produced per milligram of the substrate. In someembodiments, the decrease in the yield is less than 100%, less than200%, or less than 300% of a yield of the cannabinoid of the wild-typecannabinoid synthase or active fragment thereof. In some embodiments,the increase in the yield is more than 100%, more than 200%, or morethan 300% of a yield of the cannabinoid of the wild-type cannabinoidsynthase or active fragment thereof. In some embodiments, the variantcannabinoid synthase is a variant CBDA synthase.

In some embodiments, the non-naturally occurring amino acid sequencecomprises at least one amino acid mutation at position 69, 414, or 445relative to a wild-type consensus CBDA synthase amino acid sequence setforth in SEQ ID NO: 1046. In some embodiments, the at least one aminoacid mutation at position 69 is selected from the group consisting of:H69R, H69G, H69K, H69Q, H69A, and H69V. In some embodiments, the atleast one amino acid mutation at position 414 is selected from the groupconsisting of: A414T, A414I, and A414V. In some embodiments, the atleast one amino acid mutation at position 445 is I445V.

In some embodiments, the non-naturally occurring amino acid sequencecomprises at least two amino acid mutations at positions selected fromthe group consisting of: 69, 180, 414, and 445 relative to a wild-typeconsensus CBDA synthase amino acid sequence set forth in SEQ ID NO:1046. In some embodiments, one of the at least two amino acid mutationsis at amino acid position 69. In some embodiments, one of the at leasttwo amino acid mutations at amino acid position 69 is selected from thegroup consisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I, H69K,H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y. Insome embodiments, one of the at least two amino acid mutations at aminoacid position 69 is selected from the group consisting of H69K, H69Q,H69V, and H69G. In some embodiments, one of the at least two amino acidmutations is at amino acid position 180. In some embodiments, one of theat least two amino acid mutations at amino acid position 180 is selectedfrom the group consisting of C180A, C180D, C180E, C180F, C180G, C180H,C180I, C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C1805, C180T,C180V, C180W, and C180Y. In some embodiments, one of the at least twoamino acid mutations is at amino acid position 414. In some embodiments,one of the at least two amino acid mutations at amino acid position 414is selected from the group consisting of A414C, A414D, A414E, A414F,A414G, A414H, A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R,A414S, A414T, A414V, A414W, and A414Y. In some embodiments, one of theat least two amino acid mutations at amino acid position 414 is selectedfrom the group consisting of: A414V and A414I. In some embodiments, oneof the at least two amino acid mutations is at amino acid position 445.In some embodiments, one of the at least two amino acid mutations atamino acid position 445 is selected from the group consisting of I445A,I445C, I445D, I445E, I445F, I445G, I445H, 1445K, I445L, I445M, I445N,I445P, I445Q, I445R, I445S, I445T, I445V, 1445W, and I445Y. In someembodiments, one of the at least two amino acid mutations at amino acidposition 445 is I445M. In some embodiments, the at least two amino acidmutations are at a pair of positions selected from the group consistingof: 69/180, 69/414, 69/445, 180/414, 180/445, and 414/ 445. In someembodiments, the at least two amino acid mutations are selected from thegroup consisting of: A414V/H69K, A414V/H69Q, A414V/H69V, A414V/H69G,A414V 444M, A414I/H69K, I445M/H69K, and I445M/H69Q. In some embodiments,the non-naturally occurring amino acid sequence comprises at least threeamino acid mutations at positions selected from the group consisting of:69, 180, 414, and 445 relative to a wild-type consensus CBDA synthaseamino acid sequence set forth in SEQ ID NO: 1046. In some embodiments,the at least three amino acid mutations are at a triple of positionsselected from the group consisting of: 69/180/441, 69/180/445,69/414/445, and 180/414/445. In some embodiments, the at least threeamino acid mutations are H69Q/A414V/I445M. Disclosed herein, in certainembodiments, are variant cannabidiolic acid (CBDA) synthases or activefragments thereof comprising an amino acid mutation at a positionselected from the group consisting of: 69, 414, 180, and 445 relative toa wild-type consensus CBDA synthase set forth in SEQ ID NO: 1046.

In some embodiments, a mutation at amino acid position 69 is selectedfrom the group consisting of: H69R, H69G, H69K, H69Q, H69A, and H69V. Insome embodiments, the mutation at amino acid position 69 results in thevariant CBDA synthase producing an increase yield of a CBDA relative toa wild type CBDA synthase or active fragment thereof In someembodiments, the yield of the variant or active fragment thereof is morethan 100%, more than 200%, or more than 300% of a yield of CBDA of thewild-type CBDA synthase or active fragment thereof In some embodiments,the yield is a nMol of CBDA produced per milligram of a substrate. Insome embodiments, the substrate is cannabigerolic acid (CBGA).

In some embodiments, a mutation at amino acid position 414 is selectedfrom the group consisting of: A414T, A414I, and A414V. In someembodiments, the mutation at amino acid position 414 results in thevariant CBDA synthase producing an increase yield of a CBDA relative toa wild type CBDA synthase or active fragment thereof In someembodiments, the yield of the variant or active fragment thereof is morethan 100% or more than 200% of a yield of CBDA of the wild-type CBDAsynthase or active fragment thereof. In some embodiments, the yield is anMol of CBDA produced per milligram of a substrate. In some embodiments,the substrate is cannabigerolic acid (CBGA).

In some embodiments, a mutation at amino acid position 445 is selectedfrom the group consisting of: I445M, I445L, and I445V. In someembodiments, the mutation I445V results in the variant CBDA synthaseproducing an increase yield of a CBDA relative to a wild type CBDAsynthase or active fragment thereof. In some embodiments, the yield ofthe variant or active fragment thereof is more than 100% of a yield ofCBDA of the wild-type CBDA synthase or active fragment thereof. In someembodiments, the yield is a nMol of CBDA produced per milligram of asubstrate. In some embodiments, the substrate is cannabigerolic acid(CBGA).

In some embodiments, the mutation I445L or I445M results in the variantCBDA synthase produces an increased proportion of cannabidiolic acid(CBDA): tetrahydrocannabinolic acid (THCA) relative to a wild type CBDAsynthase or active fragment thereof In some embodiments, a proportion ofCBDA:THCA produced by the wild type CBDA synthase or active fragmentthereof is about 95:5.

Disclosed herein, in certain embodiments, are variant cannabidiolic acid(CBDA) synthases or active fragments thereof comprising at least twoamino acid mutations at amino acid positions selected from the groupconsisting of: 69, 180, 414, and 445, relative to a wild-type consensusCBDA synthase set forth in SEQ ID NO: 1046. In some embodiments, the atleast two amino acid mutations result in the variant CBDA synthaseproducing an increase yield of a CBDA relative to a wild type CBDAsynthase or active fragment thereof. In some embodiments, the yield ofthe variant or active fragment thereof is more than 100%, more than200%, more than 300%, more than 400%, more than 500%, more than 600%,more than 700%, or more than 800% of a yield of CBDA of the wild-typeCBDA synthase or active fragment thereof In some embodiments, the yieldis a nMol of CBDA produced per milligram of a substrate. In someembodiments, the substrate is cannabigerolic acid (CBGA). In someembodiments, the variant CBDA synthase or active fragment thereofproduces an increased proportion of cannabidiolic acid (CBDA):tetrahydrocannabinolic acid (THCA) relative to the wild-type CBDAsynthase or active fragment thereof. In some embodiments, a proportionof CBDA:THCA produced by the wild type CBDA synthase or active fragmentthereof is about 95:5.

In some embodiments, one of the at least two amino acid mutations is atamino acid position 69. In some embodiments, one of the at least twoamino acid mutations at amino acid position 69 is selected from thegroup consisting of H69A, H69C, H69D, H69E, H69F, H69G, H691, H69K,H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y. Insome embodiments, one of the at least two amino acid mutations at aminoacid position 69 is selected from the group consisting of H69K, H69Q,H69V, and H69G.

In some embodiments, one of the at least two amino acid mutations is atamino acid position 180. In some embodiments, one of the at least twoamino acid mutations at amino acid position 180 is selected from thegroup consisting of C180A, C180D, C180E, C180F, C180G, C180H, C1801,C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V,C180W, and C180Y.

In some embodiments, one of the at least two amino acid mutations is atamino acid position 414. In some embodiments, one of the at least twoamino acid mutations at amino acid position 414 is selected from thegroup consisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I,A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V,A414W, and A414Y. In some embodiments, one of the at least two aminoacid mutations at amino acid position 414 is selected from the groupconsisting of: A414V and A414I.

In some embodiments, one of the at least two amino acid mutations is atamino acid position 445. In some embodiments, one of the at least twoamino acid mutations at amino acid position 445 is selected from thegroup consisting of I445A, I445C, I445D, I445E, I445F, I445G, I445H,I445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T, I445V,I445W, and I445Y. In some embodiments, one of the at least two aminoacid mutations at amino acid position 445 is I445M. In some embodiments,the at least two amino acid mutations are selected from the groupconsisting of: A414V/H69K, A414V/H69Q, A414V/H69V, A414V/H69G,A414V/I44M, A414I/H69K, I445M/H69K, and I445M/H69Q.

In some embodiments, the variant CBDA synthase comprises at least threeamino acid mutations at amino acid positions selected from the groupconsisting of: 69, 180, 414, and 445, relative to a wild-type consensusCBDA synthase set forth in SEQ ID NO: 1046. In some embodiments, one ofthe at least three amino acid mutations is at amino acid position 69. Insome embodiments, one of the at least three amino acid mutations atamino acid position 69 is selected from the group consisting of H69A,H69C, H69D, H69E, H69F, H69G, H691, H69K, H69L, H69M, H69N, H69P, H69Q,H69R, H69S, H69T, H69V, H69W, and H69Y. In some embodiments, one of theat least three amino acid mutations at amino acid position 69 isselected from the group consisting of H69K, H69Q, H69V, and H69G. Insome embodiments, one of the at least three amino acid mutations is atamino acid position 180. In some embodiments, one of the at least threeamino acid mutations at amino acid position 180 is selected from thegroup consisting of C180A, C180D, C180E, C180F, C180G, C180H, C180I,C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V,C180W, and C180Y. In some embodiments, one of the at least three aminoacid mutations is at amino acid position 414. In some embodiments, oneof the at least three amino acid mutations at amino acid position 414 isselected from the group consisting of A414C, A414D, A414E, A414F, A414G,A414H, A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S,A414T, A414V, A414W, and A414Y. In some embodiments, one of the at leastthree amino acid mutations at amino acid position 414 is selected fromthe group consisting of: A414V and A414I. In some embodiments, one ofthe at least three amino acid mutations is at amino acid position 445.In some embodiments, one of the at least three amino acid mutations atamino acid position 445 is selected from the group consisting of I445A,I445C, I445D, I445E, I445F, I445G, I445H, I445K, I445L, I445M, I445N,I445P, I445Q, I445R, I445S, I445T, I445V, I445W, and I445Y. In someembodiments, one of the at least three amino acid mutations at aminoacid position 445 is I445M. In some embodiments, the at least threeamino acid mutations are H69Q, A414V, and I445M.

Disclosed herein, in certain embodiments, are nucleic acid constructscomprising a nucleic acid encoding the variant cannabinoid synthase oractive fragment thereof described herein operably linked to a promoter.Further disclosed herein, in certain embodiments, are vectors comprisingthe nucleic acid constructs described herein. Further disclosed herein,in certain embodiments, are microorganisms comprising the nucleic acidconstructs described herein. In some embodiments, the microorganism is ayeast or a bacteria. In some embodiments, the yeast is a Saccharomycescerevisiae. In some embodiments, the bacteria is an Escherichia coli.

Disclosed herein, in certain embodiments, are plants comprising thenucleic acid constructs described herein. The plant can be a vascularplant or a non-vascular plant. In some embodiments, the vascular plantis a plant in the genus Cannabis. In some embodiments, the plant isgenus Cannabis is selected from the group consisting of: Cannabissatvia, Cannabis indica, and Cannabis ruderalis. In some embodiments,the non-vascular plant is a microalgae. Disclosed herein, in certainembodiments, are methods of producing a cannabinoid, comprising: (i)contacting a cell with a nucleic acid encoding the variant cannabinoidsynthase described herein, (ii) expressing the variant cannabinoidsynthase, and (iii) isolating a cannabinoid produced by the cell. Insome embodiments, the cell is a plant cell or a microorganism cell. Insome embodiments, the method further comprises expanding the cell toproduce a plurality of expanded cells. In some embodiments, theexpanding occurs in a bioreactor. In some embodiments, the methodfurther comprises isolating and purifying the cannabinoid from theplurality of expanded cells. Further disclosed herein, in certainembodiments, are methods of producing a cannabinoid, comprising:contacting the variant cannabinoid synthase described herein to asubstrate of the variant cannabinoid synthase. In some embodiments, thecontacting occurs ex vivo. In some embodiments, the substrate is anaturally occurring substrate. In some embodiments, the naturallyoccurring substrate is selected from the group consisting ofcannabigerol (CBG), cannabigerolic acid (CBGA), cannbigerovarinic acid(GBGVA), and any homolog thereof. In some embodiments, the substrate isa non-naturally occurring substrate. In some embodiments, thenon-naturally occurring substrate can comprise: a non-naturallyoccurring tail variant, a prenyl donor, or a combination thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the compositions described herein are set forthwith particularity in the appended claims. A better understanding of thefeatures and advantages of the present disclosure will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the compositionsare utilized, and the accompanying drawings of which:

FIG. 1A shows superposition of the THCAS structure 3VTE (black) andhomology model of CBCAS (gray). The protein backbone is shown in cartoonform. The four amino acid residue changes identified (V288M, M290T,I294R, and K296R) are shown as stick figures in black for the THCASstructure, and gray for the CBCAS homology model. The flavin cofactorfrom structure 3VTE is shown in black.

FIG. 1B shows superposition of the THCAS structure 3VTE (black) andhomology model of CBDAS (gray). The protein backbone is shown in cartoonform. The active site amino acid residue substitutions (n=4) identifiedin the active site (i.e. Q69H, G180C, V415A, and T446I) are shown asstick figures in black for the THCAS structure, and gray for the CBDAShomology model. The flavin cofactor from structure 3VTE is shown ingray.

FIG. 2 shows multiple alignments of amino acid sequence for CBDAS (SEQID NO: 1043; CBDA_E55107.1), THCAS (SEQ ID NO: 1044; THCAS_E33090.1),and CBCAS (SEQ ID NO: 1045; CBCAS_JP2016). The top sequence representsthe amino acid sequence in standard IUPAC notation for SEQ ID NO: 1043.In the remainder of the alignments a dot (.) represents identity to thetop CBDAS sequence, a letter represents a substitution relative to thetop CBDAS sequence. The sequence CBDAS_AA_consensus (SEQ ID NO: 1046)represents the sequence used in the current cloning study. The signalingsequence that was used in this study is positioned on the 5′ end of thesequence and is marked in lower case standard IUPAC amino acid codes.The signaling sequence region is demarcated with white diamonds (⋄).Greyscale highlighted amino acid positions are targets for sitesaturated mutagenesis. The sequence for CBCAS (SEQ ID NO: 1045;CBCAS_JP2016) is derived from patent application US20170211049A1 (Pageand Stout 2016). Deep pocket sites are marked with solid square (▪).Outer pocket sites are marked with white square (□).

FIG. 3 shows multiple sequence alignment for parsimony informative sitesthat differentiate the CBCAS clade vs the THCAS clade (Table 15). Greyhighlight indicates sites where CBCAS harbors a derived (i.e.non-ancestral CBDAS state) substitution. Black squares (▪) indicatesites that are differentially changed relative to CBDAS both in THCASand CBCAS. These sites may have experience relaxed selection, and/or arecandidates for evolutionarily “malleable” sites. White diamonds (⋄)indicate sites that are evolutionarily conserved in the other synthases(i.e. CBDAS and THCAS), while these sites present amino acidsubstitutions in CBCAS. The evolutionary conservation of sites suggestsa functional role, so changes in CBCAS may yield unique functionaleffect. Substitutions were also categorized as those that change thetype of amino acid side chain (●) compared to substitutions that retainthe same type of amino acid side chain (o).

FIG. 4 shows neighbor joining tree for sequences from Kojoma et al.(2006), SEQ ID NO: 1052-SEQ ID NO: 1064 (Table 15), CBDA and THCA fromGenbank and CBCA from Page and Stout (2017) (Table 14). Tree was builtBy Jukes-cantor method. Ranch lengths are marked respectively.

FIG. 5A shows liquid chromatography results for a THCAS reaction. FIG.5B shows liquid chromatography results for a CBDAS reaction. FIG. 5Cshows liquid chromatography results of a standard.

FIG. 6 shows consensus reference sequence for cloning CBDAS with S.cerevisiae codon optimization (SEQ ID NO: 1049). Nucleotide sequence ismarked in bold capital letters. 1-letter amino acid translation islisted below (and in the center) of each 3-nucleotide codon. Greyscalehighlighted codons are targets for site saturated mutagenesis with theamino acid position relative to CBDAS_E55107.1 demarcated below.

FIG. 7 shows consensus reference sequence for cloning THCAS with S.cerevisiae codon optimization (SEQ ID NO: 1050). Nucleotide sequence ismarked in bold capital letters. 1-letter amino acid translation islisted below (and in the center) of each 3-nucleotide codon. Greyscalehighlighted codons are targets for site saturated mutagenesis with theamino acid position relative to THCAS_E33090.1 demarcated below.

FIG. 8 shows THCA and CBDA production profile for variants of THCAS.Relative proportion of THCA (solid grey bar) and CBDA (diagonal hatchmark bar) is depicted in each bar. The sum of the proportions of the twocannabinoids equals to unity (1.0). The respective amino acidsubstitutions for each variant are listed below the bars. The sequenceID numbers (SEQ ID NO: 321 to SEQ ID NO:335, which are equivalent toDNA_321 to DNA_335) correlating to Table 3 are labeled across the bottomof the figure. Bold labeled SEQ IDs are those variants that harbor thesubstitutions V415A and T446I, which are preferred substitutions foraltered oxidocyclization profile.

FIG. 9A shows de novo CBCA production by THCAS variants. HPLC traces forCBCA produced by select variants that display the highest detectiblelevel of CBCA production, also including respective traces for WT THCAS,WT CBDAS and CBCA standard reference.

FIG. 9B shows de novo CBCA production by THCAS variants. QuantitativeHPLC peak integration values (for area under the curve) for CBCA peakeluting at ˜18.6 minutes, corresponding to CBCA standard.

FIG. 10 shows THCA and CBDA production profile for select variants ofCBDAS. Relative proportion of CBDA (solid grey bar) and THCA (checkeredhatch mark bar) is depicted in each bar. The sum of the proportions ofthe two cannabinoids equals to unity (1.0). The respective amino acidsubstitutions for each variant are listed below the bars. The sequenceID numbers (SEQ ID NO) correlating to Table 1 are labeled across thebottom of the figure.

FIG. 11 illustrates relative activity of CBDA synthase site H69 sitesaturation mutants compared to a wild type CBDA synthase.

FIG. 12 illustrates relative activity of CBDA synthase site C180 sitesaturation mutants compared to a wild type CBDA synthase.

FIG. 13 illustrates relative activity of CBDA synthase site A414 sitesaturation mutants compared to a wild type CBDA synthase.

FIG. 14 illustrates relative activity of CBDA synthase site 1445 sitesaturation mutants compared to a wild type CBDA synthase.

FIG. 15 illustrates total products in nMol/mg enzyme generated fromCBDAS stacking variants with CBGA as a substrate.

FIG. 16 illustrates percent relative activity vs the wild type enzyme ofCBDAS stacking variants with CBGA as a substrate.

FIG. 17 mAU*min normalized to mg of protein CBDAS Stacking Variants withCBGVA as a substrate.

FIG. 18 illustrates a CBGA structure with an R group indicating thelocation of a tail variant.

FIG. 19 illustrates a CBGA structure with an R group indicating thelocation of a tail variant and the labeled positions 2-O, CO, C-5, and4-O indicating positions for attachment of a prenyl donor.

DETAILED DESCRIPTION

The primary cannabinoid components in C. sativa, affect receptorsthroughout the human body mainly by activating two specific cannabinoidreceptors (CB 1 and CB2). These receptors also bind ‘endogenous’cannabinoids (i.e. endocannabinoids), which are naturally produced bythe human body. Recent studies of the cannabinoid signaling system showsits involvement in a variety of pathological conditions. Olivetol andolivetolic acid are important intermediates in the biosynthesis of thetherapeutic plant derived polyketide-terpene natural cannabinoidproducts found in Cannabis sativa. These intermediates are prenylated bythe C. sativa endogenous prenytransferase (CsPT1) to create CBG and CBGArespectively and it is this reaction that is believed to be one of thelimiting steps in creating an engineered cannabinoid pathway in amicro-organism. CBGA is the central precursor molecule used tosynthesize most of the known phytocannabinoids in the plant. Mostphytocannabinoids require a single synthase enzyme to convert CBGA to agiven phytocannabinoid. For example, Tetrahydrocannabinolic acidsynthase, Cannabidiolic acid synthase and Cannabichromenic acid synthaseconvert CBGA to THCA, CBDA and CBCA, respectively. Many of the plantcannabinoids are non-psychoactive (e.g. CBD, CBN) and appear to havemedicinal properties but are produced by the plant at very low levels.These desirable cannabinoids are not easily separated into pure formswhen extracted from the cannabinoid plant and obtaining thesenon-abundant cannabinoids in industrial pure forms would be costprohibitive, so reconstruction of the cannabinoid pathway in amicro-organism as described herein provides the solution and the key tothe development of these cannabinoids into human therapeutics.Expressing a soluble cannabinoid synthase that is able to produce anatural or novel cannabinoid with high productivity, purity, or thecombination thereof could be critical to successfully expressing thecannabinoid plant pathway in a micro-organism for therapeuticapplications. Natural cannabinoid synthases do not exhibit high fidelityin product formation by producing a heterogeneous mixture of multiplecannabinoid molecules. For example, CBDAS produces both CBDA and THCA ata ratio of 95% to 5% respectively. Also, THCA is reported to produceboth THCA and CBCA under specific reaction conditions. Such aheterogeneous mixture of products is suboptimal for most controlledtherapeutic applications.

Described herein, in certain embodiments, are novel cannabinoidsynthases (also referred to herein as variant cannabinoid synthases) oractive fragments thereof comprising a non-naturally occurring amino acidsequence relative to a wild-type cannabinoid synthase or active fragmentthereof. In some embodiments, the variant cannabinoid synthase can acton a substrate to produce an altered amount of a cannabinoid relative toan amount of the cannabinoid produced by the wild-type cannabinoidsynthase or active fragment thereof. Altering the amount of thecannabinoid can comprise a change the ratio of the products producedrelative to the native CBDAS, THCAS and/or CBCAS cannabinoid synthaseprotein. Specifically, in some embodiments, a useful and improvedvariant cannabinoid synthase produces only a single cannabinoid productwith very high-fidelity, thus yielding a homogeneous and pure productamenable for therapeutic applications. Accordingly, provided herein insome embodiments, are variant cannabinoid synthase proteins that produce100% CBDA, or 100% THCA, or 100% CBCA or 100% of another cannabinoid.Further provided herein, in some embodiments, are variant cannabinoidsynthase proteins that produce a higher amount, or yield, ofcannabinoids relative to a wild type cannabinoid synthase protein.Further provided herein, in some embodiments, are variant cannabinoidsynthase proteins that produce a lower amount, or yield, of cannabinoidsrelative to a wild type cannabinoid synthase protein.

In some instances, to create these cannabinoid synthases, a library ofprotein variants of cannabinoid synthases can be constructed, bysynthesizing cannabinoid synthase genes and inserting rationallytargeted amino acid substitutions. These mutated cannabinoid synthasegenes can be expressed, purified and tested for the enzyme activity thatcould alter the repertoire of the respective cannabinoid synthase in adesired manner to create an enzymatic production system for natural ornovel cannabinoid molecules in micro-organisms. This cannabinoidsynthases, methods, systems and microorganisms provided herein, incertain embodiments, open up new therapeutic avenues based on theability to modulate the endocannabinoid system.

As described herein, novel cannabinoid synthase amino acid sequencesthat alter the enzyme repertoire relative to naturally occurringcannabinoid synthase (i.e. “AA_Consensus” or “native consensussequence”). In some embodiments, variant cannabinoid synthases areprovided that produce 100% CBDA; that produce 100% CBCA; and/or thatproduce other cannabinoids different from the WT parental cannabinoid.Also provided herein, in some embodiments, are novel variant cannabinoidsynthases that have improved enzyme kinetics. These un-natural variantswere initially identified in the context of single substitutionvariants, and combinations of multiple substitutions are also providedherein and these cannabinoid synthases govern the novel oxidocyclizationphenotype. These novel enzymes may be employed to exogenously expressthe cannabinoid biosynthesis pathway for large scale microbialproduction of cannabinoids.

Expressing a soluble cannabinoid synthase with specific control of basicfunctional repertoires (i.e. substrate binding, efficiency of conversionof the substrate and the oxidocyclization products profile) iscontemplated herein as useful in expressing the phytocannabinoidbiosynthesis pathway exogenously in a microorganism for industrialpurposes. This can permit the expression of desirable cannabinoids,including non-abundant phytocannabinoids, at an industrial scale and inpure form to enable development of these phytocannabinoids forapplications such as human therapeutics, nutraceuticals and otherapplications.

Other features and advantages of the cannabinoid synthases describedherein will become more readily apparent to those of ordinary skill inthe art after reviewing the following detailed description andaccompanying drawings.

Described herein, in certain embodiments, are cannabinoid synthases,nucleic acids encoding some forms of novel protein variants ofcannabinoid synthases, and various uses thereof In one embodiment,methods are provided for using site-directed mutagenesis to createlibraries and/or individual un-natural protein variants to change theprotein activity of substrate binding, efficiency of conversion of thesesubstrates, the oxidocyclization products profile specificity of thesecognate un-natural cannabinoid synthases. In another embodiment, methodsof screening these protein variants are provide to identify those whichhave altered activities and/or protein stability. In another embodiment,methods of screening compounds to identify compounds which bind to theseun-natural cannabinoid synthases and/or modulate the activity thereofare provided. In yet another embodiment, methods of screening compoundsto identify potential substrates for these un-natural cannabinoidsynthases are provided. In still another embodiment, methods areprovided for oxidocyclization of certain substrates, as well ascontrolling and/or modifying the degree of oxidocyclization promoted bythese un-natural cannabinoid synthases. In another embodiment, methodsare provided for combining the site mutations of individual proteinvariants that change general protein activity and or stability, into newcombined protein variants with enhanced general protein activity and orstability over the individual parental protein variant. In a stillfurther embodiment, methods are provided for stacking mutations, ofindividual cannabinoid synthase proteins, that change the same specificprotein activity (i.e. substrate binding, efficiency of conversion ofthese substrate, product profile of oxidocyclization products) in eachindividual protein variants into a new combined protein variant thatcombines these different mutations into one protein that furtherenhances the specific protein activity over the individual mutation andthe individual parental protein variants. In a still further embodiment,methods are provided for combining one or more novel enhanced proteinactivities (i.e. substrate binding, efficiency of conversion of thesesubstrate, profile of oxidocyclization products) together into anindividual protein variant that has many enhanced protein activities allin one protein variant, tailored to create an un-natural cannabinoidsynthase that would produce a specific group and/or individualcannabinoids.

The biochemical reaction catalyzed by THCAS is the two-electronoxidative cyclization of CBGA, catalyzed by the FAD cofactor, producingTHCA and FADH2. CBDAS catalyzes the nearly identical two-electronoxidative cyclization of CBGA by FAD, producing CBDA and FADH2.Likewise, cannabichromenic acid synthase (CBCAS) catalyzes thetwo-electron oxidative cyclization of CBGA by FAD, producing CBCA andFADH2. In all cases, the FADH2 is thought to react with molecular oxygento regenerate the FAD and produce hydrogen peroxide from oxygen. Becauseof the identical substrates and related products, it is contemplatedherein that cannabinoid produced from the transition state is determinedby the shape and electrostatic environment of the synthase's active siteand surrounding residues.

Since THCAS and CBDAS share many similarities in their biochemicalproperties, including protein mass (74 Kd), to the pI, Vmax and Km forCBGA and the amino-acid sequences being 84% identical, it is believedthat the THCAS is thought to have evolved from CBDAS by gene duplicationand divergence. Homology modeling between THCAS and CBDAS (or CBCAS) hasbeen utilized to identify unique residues in the active site and otherfunctional sites of each cannabinoid synthase for the subsequent designof mutational libraries within and around these residues coding fornon-naturally occurring variant cannabinoid synthases (e.g., variantCBDAS, THCAS or CBCAS proteins). In one embodiment, the novelcannabinoid synthases provided herein are contemplated herein to bindand catalyze the oxidocyclization of new substrates; and to controlproduct profile and the relative efficiency of each synthase.

Described herein, in certain embodiments, are five newly constructeddifferent libraries, that allow the control of the basic functionalrepertoire of the cannabinoid synthases (CBDAS, THCAS and CBCAS), suchas, e.g., controlling substrate binding, efficiency of conversion of thesubstrates and the oxidocyclization products profile. By creatinglibraries of mutations in these cannabinoid synthase proteins andscreening the libraries according to the methods provided herein,improved un-natural novel protein variants of cannabinoid synthases havebeen identified that broaden the ranges of these basic functionalaspects.

Although the native CBDAS can convert CBGA at an efficiency of 95% toCBDA, it also has an off-target conversion and produces 5% of THCA fromCBGA (FIG. 5B). This 5% THCA contamination produced by the native CBDASprevents the production of CBDA and it derivatives in a pure form, whichin turn has a negative impact on the commercialization of thesecannabinoids into a therapeutic drug from a perspective of qualitycontrol and legal regulations on THC.

To optimize CBDAS activity for utilization in an exogenousphytocannabinoid biosynthesis pathway, in particular embodiments, twolibraries of novel variants of CBDAS were created that are contemplatedherein to improve productivity of CBDA. Accordingly, these two librariesof select novel CBDAS variants have been designed to elucidate the aminoacid diversity in the active site and outer pocket of the CBDAS andelucidate novel combinations of natural mutations of native CBDAS toincrease CBDA production from CBGA by either increasing the efficiencyof oxidocyclization conversion of CBGA or increasing the product profileto >95% CBDA, with little to no THCA off targeting. In either or bothcases, these novel CBDAS variants are contemplated herein to surpass theefficiency of the native CBDAS and produce more CBDA per CBGA input.Also, in one embodiment, an improved CBDAS enzyme efficiently acceptsCBG as a substrate, (as opposed to the acid form, CBGA) to producenon-acid form cannabinoids. In this embodiment, these non-acid formcannabinoids bind more readily to human CB1 and CB2 receptors, and arepreferred for therapeutic purposes. These new CBDAS variants areunnatural variant cannabinoid synthases.

In another embodiment, provide herein is a library of select novel THCASvariants that convert the native THCAS to produce THCA from CBGA with anincrease in the efficiency of oxidocyclization conversion of CBGA, orchanging the product profile of the variant THCAS to >95% CBDA oranother cannabinoid. These THCAS variants are contemplated herein tosurpass the efficiency of the native CBDAS and producing more CBDA perCBGA input. In another embodiment, an improved THCAS enzyme efficientlyaccepts CBG as a substrate to create THC and/or CBD or anothercannabinoid. These new THCAS variants are unnatural variant cannabinoidsynthases.

In another embodiment, provided herein is a library to elucidate theamino acid diversity in the active site and outer pocket of the THCASand its effect on product profile. In this embodiment, 8 amino acidpositions have been identified in THCAS and a library is created toexplore the effect of amino acid diversity by replacing at each of these8 positions with the remaining 19 natural amino acid. These THCASvariants are contemplated herein to increase THCA production, or produceCBCA from CBGA by increasing the efficiency of oxidocyclizationconversion of CBGA. These THCAS variants are contemplated herein assurpassing the efficiency of the native THCAS and producing more THCA orCBCA per CBGA input. In other embodiments, these improved THCAS enzymeefficiently accept CBG as a substrate to create THC and/or CBC. Thesenew THCAS variants are unnatural variant cannabinoid synthases.

In yet another embodiment, provided herein is library constructed toimprove performance of CBCAS, by elucidating the amino acid diversity inn=14 selected amino acid sites in the CBCAS, chosen by evolutionaryanalysis (FIG. 3) and rational enzyme engineering. Using these selectedn=14 amino acid positions in CBCAS, a library is generated by replacingthe native amino acid at each of these n=14 positions with the remaining19 natural amino acid. These CBCAS variants are contemplated herein toincrease CBCA production by increasing the efficiency ofoxidocyclization conversion of CBGA. The specific sites are Q31, E40,P46, T74, V90, M163, A255, M288, T290, R294, L318, L391, T448, and E495.All alternative amino acids substitutions are tested at these n=14 sites(Table 5). These new CBCAS variants are unnatural variant cannabinoidsynthases.

Provided herein are novel variant cannabinoid synthases (e.g., CBDAS,THCAS and CBCAS) having an altered amino acid structure and alteredfunction compared to the respective wild-type or native consensuscannabinoid synthase (e.g., those set forth in FIG. 2, Table 14, and thelike). Specific functions to be altered may include the following:substrate binding, efficiency of conversion of the substrate, productprofile of oxidocyclization products, and the like. The cannabinoidsynthase variants allow for the production of specific small molecules(e.g., cannabinoids) that can interact with diverse biological targets;and are useful to create pharmaceutical drugs as well as chemical probesto unveil basic molecular pathways germane to health and disease.Natural products have always been an important source for newpharmaceutical drugs and controlling cannabinoid synthases providesaccess to a rich chemical diversity of novel (yet biosyntheticallynatural) molecules, thereby creating an enzymatic production system fornovel small molecules.

It is well-known that cannabinoid synthases such as the homologousenzymes, THCAS, CBDAS or CBCAS, can be defined and classified by afunctional repertoire that includes two primary components: (i)substrate utilization; and (ii) profile of products formed. Providedherein, in certain embodiments, are novel variants of these cannabinoidsynthases that have a repertoire which is significantly different thanthe native cannabinoid synthase. In particular embodiments, novelunnatural cannabinoid synthase enzymes are provided (e.g., THCAS, CBDASand CBCAS) that have improved characteristics towards optimalheterologous expression and activity of cannabinoid synthases in yeastspp., microalgae spp., and bacteria spp. The cannabinoid synthases canalso be used in transgenic plants including, but not limited to C.sativa. These improved characteristics in the cannabinoid synthasesprovided herein include, for example, increased enzymatic efficiency,improved substrate utilization, novel substrate utilization, increasedhomogeneity of oxidocyclization product, or alternatively, preferreddiversification of oxidocyclization products, and novel oxidocyclizationproduct formation for both natural and unnatural cannabinoids. In oneembodiment, one primary targeted improvement provides a novel synthasethat accepts CBG (as opposed to the acid form, CBGA) to produce non-acidform cannabinoids. These non-acid form cannabinoids bind more readily tohuman CB1 and CB2 receptors, so non-acid form cannabinoids arecontemplate herein, in one embodiment, as being preferred fortherapeutic purposes. By altering and controlling the characteristicproduct formation, the cannabinoid synthases provided herein can be usedto produce a variety of cannabinoids, either commonly found in C. sativaplants, or in low abundance in C. sativa plants, or never found in C.sativa plants. The repertoire components that govern thesecharacteristics of an variant cannabinoid synthase can be defined asfollows:

(i) Substrate utilization: Native cannabinoid synthases bind CBGA andits alkyl homologs as substrates. All the CBGA alkyl-homologs can beused as substrate and transformed by the different cannabinoid synthasesin vitro, although the efficiency of oxidocyclization conversion isknown to be different for each of the different cannabinoid synthases.The most common cannabinoids have a pentyl side-chain, but propylhomologs can also occur in vivo. Methyl-cannabinoids are also known,although these are only rarely present and typically in very smallamounts in plants. Provided herein, in certain embodiments, are panelsof novel variant cannabinoid synthases were created by mutagenesistechniques and screened for oxidocyclization by preparing appropriateenzymatic reactions with CBGA or a specific alkyl-homolog or a newunknown substrate, or CBG; and then analyzing these reactions by liquidchromatography techniques (e.g. HPLC). An oxidocyclization event issuccessful if a known or new derivative chemical species is produced orthe efficiency of conversion is changed with a known substrateenzymatically in a reaction between the novel cannabinoid synthases andsubstrate. In another embodiment, novel cannabinoid synthases areprovided that accept new substrates which native cannabinoid synthasesdo not accept, and/or change the efficiency of oxidocyclizationconversion with a known substrate relative to natural cannabinoidsynthases. The new substrate can be a naturally occurring substrate. Forexample, the naturally occurring new substrate can be naturallyoccurring CBGA or any alkyl-homolog or derivative thereof. The substratecan be a non-naturally occurring substrate. In some embodiments, thenon-naturally occurring substrate is a non-naturally occurring CBGA.

The non-naturally occurring CBGA can be a non-naturally occurringsubstrate comprising a non-naturally occurring tail variant. Thenon-naturally occurring tail variant can be a carbon chain or anaromatic ring. The tail can be branched carbon chain. The carbon chaincan comprise 1 carbon, 2 carbons, a 3 carbons, a 4 carbons, a 5 carbons,a 6 carbons, a 7 carbons, an 8 carbons, a 9 carbons, a 10 carbon s, or amore than 10 carbons. The non-naturally occurring tail variant can bethe absence of a carbon chain. In one example, the non-naturallyoccurring CBGA can be non-naturally occurring CBGA illustrated by theformula in FIG. 18, wherein the R group indicates a non-naturallyoccurring tail variant. The non-naturally occurring CBGA can comprise atleast one prenyl donor. The prenyl donor can be dimethylallyldiphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate(FPP), geranylgeranyl pyrophosphate (GGPP), or a combination thereof.The non-naturally occurring CBGA can comprise a non-naturally occurringtail variant and at least one prenyl donor. In one example, thenon-naturally occurring CBGA can be a non-naturally occurring CBGAillustrated by the formula in FIG. 19.

(ii) Profile of oxidocyclization products formed (i.e. “productprofile”): Native cannabinoid synthases, THCAS and CBCAS, naturallyyield a specific product profile for each specific substrate it binds.THCAS makes 100% THCA under standard conditions (pH 5.5) (FIG. 5a ), yetit has been reported to make a small percent of CBCA at higher pH 8-9(see, e.g., U.S. Pat. No. 9,861,609). CBCAS appears to make CBCA but, itis was previously unclear whether or not it makes some THCA in the samereaction under standard conditions, since distinguishing between THCAand CBCA by HPLC alone is not effective. In the case of CBDAS, aheterogenous mixture of CBDA and THCA is produced in a ratio of 95% CBDAto 5% THCA (FIG. 5b ) when utilizing CBGA as a substrate, under standardconditions. Under specified assay conditions (including pH, temperature,cofactor concentration, substrate, etc.), the specific oxidocyclizationproduct and the proportion of the different oxidocyclization products ispredictable and characteristic of the given conditions. Such acharacteristic oxidocyclization product or ratio of oxidocyclizationproducts is referred to herein as the cannabinoid synthase's profile ofproducts (or “product profile”). The herein reported “standard assayreaction” for THCAS and CBDAS was used to assess oxidocyclizationactivity and product profile. (The standard assay reaction was performedin a volume of 20-100 microliters and contained 20 millimolar sodiumcitrate pH 5.5, 0.2 millimolar CBGA and active cannabinoid synthaseprotein or cannabinoid synthase variant protein. These reactions wereincubated for 16 hours at 37° C.). The standard THCAS reaction yields aproduct profile that is characterized by 100% THCA (FIG. 5A) Thestandard CBDAS reaction yields a product profile that is characterized95% (±1%) CBDA and 5% (±1%) THCA (FIG. 5B). In some embodiments, novelcannabinoid synthases (e.g., CBDAS, THCAS and CBCAS) derived from thenative cannabinoid synthases are provided herein that exhibit a productprofile which is significantly different from the native cannabinoidsynthases under standard assay reaction conditions. A significantlyaltered product profile can be defined by observing any new product (asin the case of THCAS which is 100% THCA) and/or a change in a newlyproduced product in a proportion that is equal or greater than about 1%up to about 95%, about 2% up to about 85%, about 3% up to about 75%,about 4% up to about 65%, about 5% up to about 55%, about 6% up to about45%, about 7% up to about 35%, about 8% up to about 25%, about 9% up toabout 15% of total oxidocyclization product compared to the standardoxidocyclization product profile for the respective CBDAS, THCAS orCBCAS enzyme. In other embodiments, a significantly altered productprofile is defined by observing any new product and/or a change in anewly produced product in a proportion that is equal or greater thanabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30% up to about 95% of total oxidocyclization product compared to thestandard oxidocyclization product profile for the respective CBDAS,THCAS or CBCAS enzyme. In this particular embodiment, an example wouldbe the production of any CBCA or any CBDA in a THCAS standard reaction.In another embodiment, another example is a variant version of CBDAS (avariant CBDA synthase) that yields a product profile with a 4% (more orless) of CBDA and/or a 4% (less or more) of THCA. A novel productprofile may also include additional novel oxidocyclization products inthe profile mixture and is not restricted only to changes in productproportions.

A significantly altered product profile can be defined by an increase inthe yield or the decrease in the yield of at least one cannabinoid. Thecannabinoid can be a cannabichromene, a cannabicyclol, a cannbidiol, acannabielsoin, a cannabigerol, a cannabinol, a cannabinodiol, acannbitriol, a tetrahydrocannabinol, a cannbichromanon (CBCF), acannabifuran (CBF), cannabiglendol, a cannabiripsol (CBR), acannbicitran (CBT), a dehydrocannabifuran (DCBF), or any combinationthereof. The cannabichromene can be a cannabichromene (CBC),cannabichromevarin (CBCV), cannabichromenic acid (CBCA), or acombination thereof. The cannabidiol can be a cannabidiolic acid (CBDA),cannabidiol (CBD), cannabidivarin (CBDV), or a combination thereof. Thetetrahydrocannabinol can be a tetrahydrocannabinolic acid (THCA),tetrahydrocannabinol (THC), tetrahydrocannabinvarin (THCV), or acombination thereof. The increase in amount or yield can be theproduction of a cannabinoid not produced by the wild type cannabinoidsynthase.

The significantly altered product profile can be an altered amount of acannabinoid produced by the variant cannabinoid synthase. The amount,also referred to herein as the yield, can be a nMol of cannabinoidproduced per milligram of substrate. The substrate can be a naturallyoccurring substrate. The naturally occurring substrate can becannabigerol (CBG), cannabigerolic acid (CBGA), cannbigerovarinic acid(GBGVA), or any alkyl homolog or derivative thereof. The substrate canbe cannabigerol (CBG), cannabigerolic acid (CBGA), cannbigerovarinicacid (GBGVA), or any alkyl homolog thereof. The substrate can be anon-naturally occurring substrate. In some embodiments, thenon-naturally occurring substrate is a non-naturally occurring CBGA. Thenon-naturally occurring CBGA can be a non-naturally occurring CBGAcomprising a non-naturally occurring tail variant. The non-naturallyoccurring tail variant can be a carbon chain or an aromatic ring. Thetail can be branched carbon chain. The carbon chain can comprise 1carbon, 2 carbons, a 3 carbons, a 4 carbons, a 5 carbons, a 6 carbons, a7 carbons, an 8 carbons, a 9 carbons, a 10 carbon s, or a more than 10carbons. The non-naturally occurring tail variant can be the absence ofa carbon chain. In one example, the non-naturally occurring CBGA can benon-naturally occurring CBGA illustrated by the formula in FIG. 18,wherein the R group indicates a non-naturally occurring tail variant.The non-naturally occurring CBGA can further comprise at least oneprenyl donor. The prenyl donor can be dimethylallyl diphosphate (DMAPP),geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranylpyrophosphate (GGPP), or a combination thereof. In one example, thenon-naturally occurring CBGA can be a non-naturally occurring CBGAillustrated by the formula in FIG. 19.

In some embodiments, the yield of the cannabinoid produced by thevariant cannabinoid synthase is more than 100% of the yield produced bya wild-type cannabinoid synthase. In some embodiments, the yield of thecannabinoid produced by the variant cannabinoid synthase is less than100% of the yield of the cannabinoid produced by the produced by awild-type cannabinoid synthase. In some embodiments, the yield of thecannabinoid produced by the variant cannabinoid synthase is more than200% of the yield of the cannabinoid produced by the produced by awild-type cannabinoid synthase. In some embodiments, the yield of thecannabinoid produced by the variant cannabinoid synthase is less than200% of the yield produced of the cannabinoid produced by the by awild-type cannabinoid synthase. In some embodiments, the yield of thecannabinoid produced by the variant cannabinoid synthase is more than300% of the yield of the cannabinoid produced by the produced by awild-type cannabinoid synthase. In some embodiments, the yield of thecannabinoid produced by the variant cannabinoid synthase is less than300% of the yield of the cannabinoid produced by a wild-type cannabinoidsynthase. In some embodiments, the yield of the cannabinoid produced bythe variant cannabinoid synthase is more than 100%, more than 200%, morethan 300% more than 400%, more than 500%, more than 600%, more than700%, or more than 800% of the yield of the cannabinoid produced by theproduced by a wild-type cannabinoid synthase. In some embodiments, theyield of the cannabinoid produced by the variant cannabinoid synthase isless than 100%, less than 200%, less than 300% less than 400%, less than500%, less than 600%, less than 700%, or less than 800% of the yield ofthe cannabinoid produced by a wild-type cannabinoid synthase. Thealtered amount of a cannabinoid produced by the variant cannabinoidsynthase can comprise production of any amount of the cannabinoid if thecannabinoid is not produced by the wild-type cannabinoid synthase.

Site-directed mutagenesis of the amino acid residues in the nativecannabinoid synthase can be employed to advantageously alter the basicfunctional repertoire of the cannabinoid synthases, including, forexample, substrate binding, efficiency of conversion of thesesubstrates, the oxidocyclization products profile, and the like. Bycreating libraries of mutations in the cannabinoid synthase proteins,un-natural novel protein variants of cannabinoid synthases can beprovided that broaden the ranges of these basic functional aspects ofcannabinoid synthase and allow for the control of oxidocyclization; andvariant cannabinoid synthases are provided that produce specificcannabinoids or novel cannabinoids never enzymatically produced before.

The enzyme THCA synthase (THCAS) and CBDA synthase (CBDAS) can beseparately cloned, heterologously expressed in microorganisms such asyeast, microalgae, and bacteria, and can produce THCA and CBDA from CBGAvia oxidative cyclization. CBCAS has received less treatment in peerreviewed literature. At a protein level the sequences for CBDAS andCBCAS are very similar to that of THCAS (>80% identity). Within theactive site of these synthases, the protein sequences are even moresimilar. Given that the same single molecule substrate (CBGA or an alkylhomolog) is used by any of these cannabinoid synthase enzymes to producetheir diverse products, homology-based modeling can be utilized toidentify residues in the “deep active site” and in the “outer pocket”that could (i) control oxidocyclization product profile of the specificsubstrate used, (ii) the kinetics and/or efficiency of oxidocyclizationconversion seen with that specific substrate and (iii) the binding of anew substrate (or multiple substrates) to create a novel cannabinoid ormultiple cannabinoids. Homology-based modeling of CBDAS and CBCAS can beconducted using the Swiss-MODEL server (https://swissmodel.expasy.org/),the source coordinates were derived from the structure of THCAS, theonly know cannabinoid synthase crystal structure solved to date, PDB ID3VTE.

The active site of CBDAS is more divergent from THCAS compared to thedivergence between CBCAS and THCAS. Based on this homology-based proteinmodeling, the active site of CBDAS was shown to exhibit four amino aciddifferences from THCAS in the “deep active site” near the catalytic FADcofactor (FIG. 1). [Note: A multiple alignment of the complete aminoacid sequences between CBDAS and THCAS displays an amino acidinsertion/deletion event at alignment position 253. Accordingly, THCAS(and CBCAS) displays a serine (S) insertion at alignment site 253 (seeFIG. 2). It follows that all homologous amino acid positions>=253through 544 on CBDAS, respectively correlate to the homologouspositions>=254 through 545 on THCAS and CBCAS] The identified divergentamino acids in the “deep active site” are as follows (according tostandard IUPAC notation, the CBDAS residue is listed first, the CBDASamino acid position is listed second, while the homologous THCAS residueis listed last): H69Q, C180G, A414V (homologous to amino acid position415 in THCAS), and I445T (homologous to amino acid position 446 inTHCAS) (FIG. 1). It is contemplated herein that several of these aminoacid substitutions can produce significant changes to the active siteshape and electrostatic environment and alter product resolution fromthe transition state.

In another embodiment, four additional changes were also identified inthe “outer pocket” of CBDAS relative to THCAS. These are contemplatedherein to impact kinetics or product identity by alteration of proteinflexibility. In this embodiment, the positions of these substitutions onCBDAS are M256I, R295K, Q376K, and N377K. (CBDA residue is listed first,while THCAS residue listed second). One of these (R295K) is the same inCBDAS and CBCAS described above.

Because of the high sequence similarity between THCAS, CBCAS, and CBDAS,it is contemplated herein, in some embodiments, that generating aminoacid diversity via site directed mutagenesis, control can be achievedof: (i) product profile of the specific substrate/s used, (ii) thekinetics and efficiency of oxidocyclization conversion seen withspecific substrate/s (iii) the binding of a new substrate to create anovel cannabinoid. Rational model-based design has identified residuesin THCAS relative to CBCAS and relative to CBDAS. In particularembodiments, altering all or some subset of these residues can producenovel variant enzymes divergent from THCAS that: 1) produces CBCA, CBDA,or another non-THCA cannabinoid from CBGA and its alkyl-homologs; 2)produces a novel variant of CBDAS that produces THCA, CBCA or anothernon-CBDA cannabinoid; and/or 3) produces a novel variant of CBCAS thatproduces CBDA, THCA or another non-CBCA cannabinoid. See FIGS. 1A & 1B.

Since no explicit sequence for CBCAS was identified in the completedgenome of Purple Kush and no CBCAS accession is currently available inGenbank, the sequence identity of this synthase appears to be unclear inthe peer-reviewed literature. Only a recent patent application by Pageand Stout (2017; US 2017/0211049 A1) proposed a sequence as shown inFIG. 2. This putative CBCAS sequence exhibits 92.6% amino acid identityto THCAS. In a different study, a set of ambiguously classifiedcannabinoid synthase DNA sequences have been reported in Kojoma et al(Forensic Sci Int. 2006 Jun 2;159(2-3):132-40. Epub 2005 Sep 6.). Kojomaet al. identifies a series of diverged sequences extracted fromdifferent C. sativa strains with ˜93.0% identity to other knownfunctional THCAS sequences. Phenotypic data from the respective strainssuggested a lack of THCA producing activity. These sequences are knownas “Kojoma-type THCAS” sequences and are classified as “defective THCASalleles”. These sequence accessions were collected and a multiplesequence alignment between CBCAS_JP2016 and these sequences from Kojomaet al. revealed 99.0%-99.4% identity between CBCAS_JP2016 and“Kojoma-type THCAS” sequences (FIG. 3). Accordingly, it is contemplatedherein that the sequences commonly referred to as “Kojoma-type THCAS”sequences are in fact likely to be CBCAS sequences, rather thandefective THCAS sequences. Provided herein, in certain embodiments, is aproposed relationship between the sequences that can be described in aphylogenetic tree, demonstrating 3 clades for the three differentsynthases (CBDAS, THCAS and CBCAS) (FIG. 4). From the multiple putativealleles of CBCAS, the amino acid substitutions that are unique to theCBCAS clade are provided herein in accordance as set forth in FIG. 3.These clade-specific amino acid substitution sites (n=25) are providedherein as candidates for amino acid sites which are important and uniquefor this specific synthase function. The amino acid substitutionscontemplated herein can be further down-selected to have an effect byreferring to homology-based modeling and/or selecting substitutions thatchange the type of amino acid side chain. In one embodiment, providedherein is a subset of n=14 sites that are contemplated herein to have aneffect on CBCAS function: Q31, E40, P46, T74, V90, M163, A255, M288,T290, R294, L318, L391, T448, E495. (Italics represents substitutionselected based on homology-based modeling, underline represents siteselected both by comparative evolution and by homology-based modeling,while normal font represents candidates only by comparative evolutionanalysis). Also provided herein are libraries for each of these sites toidentify the effect on the CBCAS repertoire for purpose of improvementof industrial biotechnological processes (Table 5).

In some embodiments, a novel cannabinoid synthase (CBDAS) can be createdthat alters the ratio of the oxidocyclization products. The wild typeCBDAS standard oxidocyclization assay with CBGA produces two products asdetermined by HPLC (15 min), with retention times of approximately 8.84minutes (an “early product” which is cannabidiolic acid [CBDA]) and11.46 minutes product which is tetrahydrocannabinolic acid (a “lateproduct”) in a ratio of 95% CBDA to 5% THCA FIG. 5b ). The 160 CBDASvariants (e.g.,“Cannabinoid synthase variants” or variant CBDAsynthases) are contemplated herein to produce a spectrum of ratiosbetween CBDA and THCA, or other products. Some variants are contemplatedherein to create a greater majority of CBDA and other variants produce asignificant greater majority of THCA. In one embodiment, novel variantCBDA synthases are provided herein that alter the ratio ofoxidocyclization products in the overall oxidocyclization profilecreated from CBDAS and using CBGA as a substrate.

It another embodiment, general regions of the protein are altered inorder to achieve desired oxidocyclization products from CBGA, andspecific amino acid residues to alter are identified to achieve desiredoxidocyclization products.

E) Individual Select Substitutions are Combined with Other SelectIndividual Mutation/s to Further Increase the Specific ProteinActivities.

A specific single amino acid substitution that significantly alters theenzyme's functional repertoire is referred to herein as a “preferredsubstitution.” In this embodiment, a novel preferred variant thatharbors a single amino acid substitution exhibits a preferred enzymefunctional repertoire is further assayed. Multiple preferredsubstitutions are combined (“stacked”) one with another substitutioninto a doubleton substitution CBDAS variant to create a combinedimproved effect for further altering and enhancing the variant's enzymerepertoire. A variant enzyme with multiple preferred substitutions iscontemplated herein to contain two or more substitutions that affectsthe same or different component/s of the repertoire (i.e., substrateutilization, product profile).

To further characterize and compare these variant cannabinoid synthaseenzymes vs WT cannabinoid synthase, oxidocyclization reaction rates areassessed for the novel cannabinoid synthase variants by employing anenzyme kinetics experimentation. Standard cannabinoid synthaseoxidocyclization assays (see above) are prepared for WT cannabinoidsynthase and select amino acid substitution variants. Oxidocyclizationprofiles are measured along a time course spanning from 0 to 20 hours ofincubation time with varying CBGA concentrations. At each time point, a20 uL standard oxidocyclization assay is extracted by ethyl acetatemethod as described above. To quantify the production of CBDA, THCA, orother cannabinoid at each timepoint, the area under the curve on eachHPLC trace is calculated for each peak representing CBDA, THCA, or othercannabinoid (mAU), respectively. Enzyme concentration is estimated bywestern blot, and by utilizing the product production rate data as afunction of substrate concentration, nonlinear curve fitting to theMichaelis-Menten equation will yield Km and Kcat values for each variantenzyme.

Different single specific amino acid substitutions are contemplatedherein to alter different aspects of the cannabinoid synthase functionalrepertoire. For example, several variant amino acid positions or singlevariant amino acid substitutions could individually, or when combined,alter substrate acceptance in some instances, thus allowing novelsubstrate usage. Such cannabinoid synthases with these single orcombined amino acid substitutions are provided herein (e.g., a variantCBDAS, THCAS or CBCAS). The novel substrate can be a non-naturallyoccurring substrate or can be a naturally occurring substrate previouslynot accepted by the cannabinoid synthase.

In another embodiment, several variant amino acid positions or singlevariant amino acid substitutions could individually, or when combined,change the products generated in an oxidocyclization reaction. In oneembodiment, a variant amino acid substitution or substitutions iscontemplated herein to cause a variant enzyme to create a product orproducts that the native cannabinoid synthase does not create. Theseproduct/s could be novel or otherwise naturally occurring from othercannabinoid synthases. Such cannabinoid synthases with these single orcombined amino acid substitutions are provided herein (e.g., a variantCBDAS, THCAS or CBCAS). In another embodiment, several variant aminoacid positions or single variant amino acid substitutions couldindividually, or when combined, alter the ratio of the oxidocyclizationproducts (e.g., the ratio of CBDA:THCA, such as 100% CBDA/0% THCA, andthe like). These cannabinoid synthases with these single or combinedamino acid substitutions are provided herein (e.g., a variant CBDAS,THCAS or CBCAS).

For example, provided here are variant CBDA synthases, wherein saidvariant is capable of shifting the ratio of CBDA to THCA (CBDA/THCA) tofavor production of CBDA (FIG. 8) compared to native consensus CBDAsynthase (SEQ ID NO. 1045) or compared to native consensus THCA synthase(SEQ ID NO. 1046). Also provide herein are variant CBDA synthases,wherein said variant is capable of shifting the ratio of THCA to CBDA(THCA/CBDA) to favor production of THCA (FIG. 10) compared to nativeconsensus CBDA synthase or compared to native consensus THCA synthase.Also provided herein are variant THCA synthases, wherein said variant iscapable of shifting the ratio of CBDA to THCA (CBDA/THCA) to favorproduction of CBDA compared to native consensus THCA synthase. Alsoprovided herein are variant THCA synthases, wherein said variant iscapable of shifting the ratio of THCA to CBDA (THCA/CBDA) to favorproduction of THCA compared to native consensus CBDA synthase orcompared to native consensus THCA synthase. Also provided herein arevariant CBCA synthases, wherein said variant is capable of shifting theratio of CBCA to THCA (CBCA/THCA) to favor production of CBCA comparedto native consensus THCA synthase. Also provided herein are variant CBCAsynthases, wherein said variant is capable of shifting the ratio of THCAto CBCA (THCA/CBCA) to favor production of THCA compared to nativeconsensus CBCA synthase or compared to native consensus THCA synthase.

As used herein, the phrase “variant is capable of shifting the ratio ofCBDA to THCA (CBDA/THCA) to favor production of CBDA” refers to anycannabinoid synthase variant described herein (e.g. CBDAS, THCAS orCBCAS) that produces a higher percentage of CBDA compared to nativeconsensus CBDA synthase, or in some embodiments when compared to nativeconsensus THCA synthase, using the standard HPLC assay described herein.

As used herein, the phrase “variant is capable of shifting the ratio ofTHCA to CBDA (THCA/CBDA) to favor production of THCA” refers to anycannabinoid synthase variant described herein (e.g. CBDAS, THCAS orCBCAS) that produces a higher percentage of THCA compared to nativeconsensus THCA synthase, or in some embodiments when compared to nativeconsensus CBDA synthase, using the standard HPLC assay described herein.

As used herein, the phrase “variant is capable of shifting the ratio ofCBCA to THCA (CBCA/THCA) to favor production of CBDA” refers to anycannabinoid synthase variant described herein (e.g. CBDAS, THCAS orCBCAS) that produces a higher percentage of CBCA compared to nativeconsensus CBCA synthase, or in some embodiments when compared to nativeconsensus THCA synthase, using the standard HPLC assay described herein.

Described herein, in certain embodiments, are variant CBDA synthase(CBDAS) comprising any combination of one up to all 8 of variant aminoacid positions set forth in Table 1, A-H, corresponding to amino acidpositons 69, 180, 414, 445, 256, 295, 376 and 377, respectively relativeto native consensus CBDA synthase set forth in FIG. 6 (SEQ ID NO: 1049),wherein each amino acid variant is selected from the group consisting ofall amino acid variants set forth in Table 1, A-H. In some embodiments,said variant comprises a variant amino acid at a number ofvariant-positions set forth in Table 1, A-H compared to native consensusCBDA synthase, wherein the number of variant-positions is selected fromthe group consisting of: 1, 2, 3, 4, 5, 6, 7 and 8. In some embodiments,the variant comprises any combination of one or more selected from thegroup consisting of: Table 1A position 69 is H69Q; Table 1B position 180is C180G; Table 1C position 414 is A414V, Table 1D position 445 isI445T; Table 1E position 256 is M256I; Table 1F position 295 is R295K,Table 1G position 376 is Q376K; and Table 1H position 377 is N377K.

In some embodiments, said variant comprises 4 variant amino acids at 4variant-positions set forth in Table 1, A-H compared to native consensusCBDA synthase, wherein each amino acid variant is selected from thegroup consisting of all amino acid variants set forth in Table 1, A-H.In some embodiments, said 4 variant positions correspond to Table 1Aposition 69; Table 1B position 180; Table 1C position 414, and Table 1Dposition 445. In some embodiments, the variant comprises any combinationof one or more selected from the group consisting of: Table 1A position69 is H69Q; Table 1B position 180 is C180G; Table 1C position 414 isA414V, and Table 1D position 445 is I445T. In some embodiments, thevariant comprises the variant amino acids corresponding to: Table 1Aposition 69 is H69Q; Table 1B position 180 is C180G; Table 1C position414 is A414V, and Table 1D position 445 is I445T. In some embodiments,said variant comprises 2 variant amino acids at 2 variant-positions setforth in Table 1, A-H compared to native consensus CBDA synthase,wherein each amino acid variant is selected from the group consisting ofall amino acid variants set forth in Table 1, A-H. In some embodiments,said 2 variant positions correspond to Table 1C position 414, and Table1D position 445. In some embodiments, the variant comprises anycombination of one or more selected from the group consisting of: Table1C position 414 is A414V and Table 1D position 445 is I445T. In someembodiments, the variant comprises the variant amino acids correspondingto: Table 1C position 414 is A414V and Table 1D position 445 is I445T.

Described herein, in certain embodiments, are variant cannabidiolic acid(CBDA) synthases or active fragments thereof comprising an amino acidmutation at a position selected from the group consisting of: 69, 414,180, and 445 relative to a wild-type consensus CBDA synthase set forthin SEQ ID NO: 1046. The amino acid mutation can be any non-naturallyoccurring amino acid relative to SEQ ID NO: 1046. The mutation canproduce an increase yield of a CBDA relative to a wild type CBDAsynthase. The increase in yield can be an increase in yield of more than100%, more than 200%, or more than 300%. The mutation producing theincrease in yield can be: a mutation at amino acid position 69 selectedfrom the group consisting of: H69R, H69G, H69K, H69Q, H69A, and H69V, amutation at amino acid position 414 selected from the group consistingof A414T, A414I, and A414V, or a mutation at amino acid position 445selected from I445V. The mutation can produce a change in ratio of afirst cannabinoid to a second cannabinoid. In one example, a variantCBDA synthase comprising a mutation selected from the group consistingof I445M and I445L can produce an increase in a ratio of CBDA:THCArelative to a wild type CBDA synthase.

In some embodiments, said variant comprises a variant amino acid at 1variant-position set forth in Table 1, A-H compared to native consensusCBDA synthase, wherein said amino acid variant is selected from thegroup consisting of all amino acid variants set forth in Table 1, A-H.In some embodiments, said variant position corresponds to a variantposition selected from the group consisting of: Table 1C position 414,Table 1D position 445, Table IF position 295. In some embodiments, saidvariant position corresponds to a variant position selected from thegroup consisting of: Table 1C position 414 is A414V; Table 1D position445 is I445T; and Table IF position 295 is R295K.

In some embodiments, said variant is capable of shifting the ratio ofCBD to THC (CBD/THC) to favor production of CBD (FIG. 8) compared tonative consensus CBDA synthase or compared to native consensus THCAsynthase.

Further described herein, in certain embodiments, are variant THCAsynthase (THCAS) comprising any combination of one up to all 8 ofvariant amino acid positions set forth in Table 2, A-H, corresponding toamino acid positons 69, 180, 415, 446, 257, 296, 377 and 378,respectively relative to native consensus THCA synthase set forth inFIG. 2, wherein each amino acid variant is selected from the groupconsisting of all amino acid variants set forth in Table 2, A-H. In someembodiments, said variant comprises a variant amino acid at a number ofvariant-positions set forth in Table 2, A-H compared to native consensusTHCA synthase, wherein the number of variant-positions is selected fromthe group consisting of: 1, 2, 3, 4, 5, 6, 7 and 8. In some embodiments,the variant comprises any combination of one or more selected from thegroup consisting of: Table 2A position 69 is Q69H; Table 2B position 180is G180C; Table 2C position 415 is V415A, Table 2D position 446 isT446I; Table 2E position 257 is I257M; Table 2F position 296 is K296R,Table 2G position 377 is K377Q; and Table 2H position 378 is K378N.

In some embodiments, said variant comprises 4 variant amino acids at 4variant-positions set forth in Table 2, A-H compared to native consensusTHCA synthase, wherein each amino acid variant is selected from thegroup consisting of all amino acid variants set forth in Table 2, A-H.In some embodiments, said 4 variant positions correspond to Table 2Aposition 69; Table 2B position 180; Table 2C position 415, and Table 2Dposition 446. In some embodiments, the variant comprises any combinationof one or more selected from the group consisting of: Table 2A position69 is Q69H; Table 2B position 180 is G180C; Table 2C position 415 isV415A, and Table 2D position 446 is T446I. In some embodiments, thevariant comprises the variant amino acids corresponding to: Table 2Aposition 69 is Q69H; Table 2B position 180 is G180C; Table 2C position415 is V415A, and Table 2D position 446 is T446I.

In some embodiments, said variant comprises 2 variant amino acids at 2variant-positions set forth in Table 2, A-H compared to native consensusTHCA synthase, wherein each amino acid variant is selected from thegroup consisting of all amino acid variants set forth in Table 2, A-H.In some embodiments, said 2 variant positions correspond to Table 2Cposition 415, and Table 2D position 446. In some embodiments, thevariant comprises any combination of one or more selected from the groupconsisting of: Table 2C position 415 is V415A and Table 2D position 446is T446I. In some embodiments, the variant comprises the variant aminoacids corresponding to: Table 2C position 415 is V415A and Table 2Dposition 446 is T446I.

In some embodiments, said variant comprises a variant amino acid at 1variant-position set forth in Table 2, A-H compared to native consensusTHCA synthase, wherein said amino acid variant is selected from thegroup consisting of all amino acid variants set forth in Table 2, A-H.In some embodiments, said variant position corresponds to a variantposition selected from the group consisting of: Table 2C position 415,Table 2D position 446, Table 2F position 296. In some embodiments, saidvariant position corresponds to a variant position selected from thegroup consisting of: Table 2C position 415 is V415A; Table 2D position446 is T445I; and Table 2F position 296 is K296R.

In some embodiments, said variant is capable of shifting the ratio ofCBD to THC (CBD/THC) to favor production of CBD compared to nativeconsensus THCA synthase.

Further described herein, in certain embodiments, are variant THCAsynthase relative to native consensus THCA synthase sequence set forthin FIG. 2, wherein the variant THCA synthase is selected from the groupof variants set forth in Table 3. Also descirbed herein, are variantCBDA synthase relative to native consensus CBDA synthase sequenceCBDAS_E55107.1 set forth in FIG. 6, wherein the variant CBDA synthase isselected from the group of variants set forth in Table 4.

Described herein, in certain embodiments, are variant CBDA synthase(CBDAS) relative to native consensus CBDA synthase sequenceCBDAS_E55107.1 set forth in FIG. 6, wherein the variant CBDA synthasecomprises any combination of one up to 6 variant amino acids atpositions set forth in Table 4, corresponding to amino acid positons 74,143, 168, 196, 474 and 543, wherein each amino acid variant is selectedfrom the 19 other natural amino acids different from the native aminoacid in FIG. 6. In some embodiments, said variant comprises a variantamino acid at a number of variant-positions set forth in Table 4compared to native consensus CBDA synthase, wherein the number ofvariant-positions is selected from the group consisting of: 1, 2, 3, 4,5, and 6. In some embodiments, the variant comprises any combination ofone or more selected from the Table 4 group consisting of: T74S, H143R,N168S, N196S, K474Q, and R543H. In some embodiments, the variant furthercomprises any combination of one up to all 8 of variant amino acidpositions set forth in Table 1, A-H, corresponding to amino acidpositons 69, 180, 414, 445, 256, 295, 376 and 377, respectively relativeto native consensus CBDA synthase set forth in FIG. 6, wherein eachamino acid variant at positons 69, 180, 414, 445, 256, 295, 376 and 377is selected from the group consisting of all amino acid variants setforth in Table 1, A-H, respectively. In some embodiments, said variantcomprises a variant amino acid at a number of variant-positions setforth in Table 1, A-H and Table 4 compared to native consensus CBDAsynthase, wherein the number of variant-positions is selected from thegroup consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.In some embodiments, the variant comprises any combination of one ormore selected from the group consisting of: Table 1A position 69 isH69Q; Table 1B position 180 is C180G; Table 1C position 414 is A414V,Table 1D position 445 is I445T; Table 1E position 256 is M256I; Table 1Fposition 295 is R295K, Table 1G position 376 is Q376K; and Table 1Hposition 377 is N377K; Table 4 T74S; Table 4 H143R; Table 4 N168S; Table4 N196S; Table 4 K474Q; and Table 4 R543H.

Described herein, in certain embodiments, are variant CBCA synthase(CBCAS) relative to native consensus CBCA synthase sequence CBCAS_JP2016set forth in FIG. 2, wherein the variant CBCA synthase comprises anycombination of one up to all 14 variant amino acid positions in Table 5corresponding to amino acid positons Q31, E40, P46, T74, V90, M165,A255, M288, T290, R294, L318, L391, T448, E495, wherein each amino acidvariant at positons 31, 40, 46, 74, 90, 163, 255, 288, 290, 294, 318,391, 448 and 495 is selected from the group consisting of all amino acidvariants set forth in Table 5, respectively. In some embodiments, thenumber of variant-positions is selected from the group consisting of: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.

Described herein, in certain embodiments, are variant cannabinoidsynthase comprising any combination of variant amino acid changes amongall polypeptides set forth in Tables 1-5, relative to the respectivenative cannabinoid synthase set forth in FIG. 2, wherein the amino-acidvariants are selected from a combination of a subset of variant aminoacids among all variant-positions in polypeptides set forth in Tables1-5 and conservative substitutions at any amino acid position withinwild-type cannabinoid synthase, so long as the variant cannabinoidsynthase differs from wild-type cannabinoid synthase by 1 up to 50 aminoacids or more. In some embodiments, said variant is capable of shiftingthe ratio of THC to CBD (THC/CBD) to favor production of THC (FIG. 10)compared to native consensus CBDA synthase or compared to nativeconsensus THCA synthase. In some embodiments, the variant amino acid isat a position selected from the group consisting of: 180, 414 and 445.In some embodiments, the variant amino acid is selected from the groupconsisting of: C180L, C180R, A414C, A414F, A414H and 1445S.

In another embodiment, any one or more of the mutations (variant aminoacids at a respective variant position) from any of the “Cannabinoidsynthase variants” described herein, can be combined to look for newoxidocyclization profiles, or new ratios between oxidocyclizationproducts (i.e., the ratio between the “early product” and the “lateproduct.”). These mutations include any combination of any of themutations in the “Cannabinoid synthase library,” including any onemutation, including any combination of 2 mutations, including anycombination of 3 mutations, including any combination of 4 mutations,and including any combination of any number of mutations found in the“Cannabinoid synthase variants.”

Described herein, in certain embodiments, are variant cannabidiolic acidsynthases or active fragments thereof comprising at least 2 amino acidmutations compared to a wild-type cannabidiolic acid synthase or activefragments thereof. The variant cannabidiolic acid synthase comprisingthe at least 2 amino acid mutations can be a CBDAS, THCAS, or CBCAS. Thevariant cannabidiolic acid synthase comprising at least 2 amino acidmutations or active fragment thereof can produce an alteredoxidocyclization profile compared to a wild-type cannabidiolic acidsynthase or active fragment thereof. For example, the variantcannabidiolic acid synthase comprising at least 2 amino acid mutationsor active fragment thereof can act on a substrate to produce an alteredamount of a cannabionid relative to an amount of a cannabinoid producedby the wild-type cannabionid synthase or active fragment thereof. Thealtered amount can be an increase relative to the amount produced by thewild-type cannabinoid synthase, or a decrease relative to the amountproduced by the wild-type cannabinoid synthase. The altered amount canproduce a change in proportion of a first cannabinoid relative to asecond cannabinoid. The increase in amount can be the production of acannabinoid not produced by the wild type cannabinoid synthase.

A variant CBDAS comprising at least two amino acid mutations cancomprise a first mutation at position 69, 414, 180, or 445 relative to aconsensus CBDA synthase set forth in SEQ ID NO: 1046. The variant CBDAScomprising at least two amino acid mutations can further comprise asecond mutation at position 69, 414, 180, or 445 relative to a consensusCBDA synthase set forth in SEQ ID NO: 1046. The variant CBDAS comprisingat least two amino acid mutations can further comprise a third mutationat position 69, 414, 180, or 445 relative to a consensus CBDA synthaseset forth in SEQ ID NO: 1046. In some embodiments, the at least twoamino acid mutations can comprise a first mutation at position 256, 295,376, or 377 relative to a consensus CBDA synthase set forth in SEQ IDNO: 1046.

In some embodiments, one of the at least two amino acid mutations is atposition 69. At least one of the two mutations can be H69A, H69C, H69D,H69E, H69F, H69G, H69I, H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S,H69T, H69V, H69W, or H69Y. At least one of the two mutations can beH69K, H69Q, H69V, or H69G. In some embodiments, one of the at least twoamino acid mutations is at position 180. At least one of the twomutations can be C180A, C180D, C180E, C180F, C180G, C180H, C180I, C180K,C180L, C180M, C180N, C180P, C180Q, C180R, C1805, C180T, C180V, C180W, orC180Y. In some embodiments, one of the at least two amino acid mutationsis at position 414. At least one of the two mutations can be A414C,A414D, A414E, A414F, A414G, A414H, A414I, A414K, A414L, A414M, A414N,A414P, A414Q, A414R, A4145, A414T, A414V, A414W, and A414Y. At least oneof the two mutations can be A414V or A414I. In some embodiments, one ofthe at least two amino acid mutations is at position 445. At least oneof the two mutations can be I445A, I445C, I445D, I445E, I445F, I445G,I445H, I445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T,I445V, I445W, or I445Y. At least one of the two mutations can be I445M.

In some embodiments, the at least two amino acid mutations are at a pairof positions selected from the group consisting of: 69/180, 69/414,69/445, 180/414, 180/445, or 414/445. The at least two amino acidmutations can be A414V /H69K, A414V /H69Q, A414V/H69V, A414V /H69G,A414V 444M, A414I/H69K, I445M/H69K, or I445M/H69Q. In some embodiments,the at least three amino acid mutations are at a triple of positionsselected from the group consisting of: 69/180/441, 69/180/445,69/414/445, and 180/414/445. The at least three amino acid mutations canbe H69Q/A414V/I445M.

Described herein, in certain embodiments, are nucleic acid constructsencoding a variant cannabinoid synthase described herein. The nucleicacid construct can comprise a nucleic acid encoding the variantcannabinoid synthase operably linked to a promoter. The nucleic acidconstruct can be a DNA or an RNA. Further described herein, in certainembodiments, are vectors comprising the nucleic acid constructsdescribed herein. In some embodiments, the vector is a viral vector or anon-viral vector. In some embodiments, the vector is a viral vector. Insome embodiments, the viral vector is a retroviral vector, an adenoviralvector, an adeno associated virus (AAV) vector, an alphavirus vector, avaccinia virus vector, a herpes simplex virus (HSV) vector, a lentivirusvector, or a retrovirus vector. In some embodiments, the viral vector isan adeno associated virus (AAV) vector. In some embodiments, the adenoassociated viral vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, orAAV8. In some embodiments, the viral vector is a replication-competentviral vector or a replication-incompetent viral vector. In someembodiments, the non-viral vector is a plasmid, a naked nucleic acid, ornucleic acid complexed with a delivery vehicle. In some embodiments, theplasmid is complexed with a delivery vehicle. In some embodiments, thedelivery vehicle is a lipid. In some embodiments, the lipid is aliposome.

Further described herein, in certain embodiments, are microorgansimscomprising the nucleic acid constructs described herein. Themicroorganism can be a yeast. The yeast can be a Saccharomycescerevisiae. The microorganism can be a bacterium. The bacterium can bean Escherichia coli. The microorganism can comprise a vector comprisinga nucleic acid construct encoding a variant cannabinoid synthasedescribed herein.

Further described herein, in certain embodiments, are plants comprisingthe nucleic acid constructs encoding the variant cannabinoid synthasesdescribed herein. The plant can be a vascular plant. The vascular plantcan be a plant in the family Cannabaceae. The plant can be a plant inthe genus Cannabis. The plant in the genus Cannibis can be a plantselected from the group consisting of Cannabis satvia, Cannabis indica,and Cannabis ruderalis. The plant can be a non-vascular plant. Thenon-vascular plant can be an algae. The algae can be a microalgae. Thenucleic acid encoding a variant cannabinoid synthase can be integratedinto the genome of the plant. The nucleic acid encoding a variantcannabinoid synthase can be a vector.

Further described herein, in certain embodiments, are recombinantmethods of producing a variant cannabinoid synthase comprisingexpressing the nucleic acid constructs described herein. In someembodiments, producing a variant cannabinoid synthase comprise: (i)contacting a cell with a nucleic acid construct encoding the variantcannabinoid synthase, and (ii) expressing the variant cannabinoidsynthase in the cell. The contacting can occur in vivo. The contactingcan occur ex vivo. In some embodiments, the method comprises expandingthe cell to produce a plurality of expanded cells. In some embodiments,the expanding occurs in a bioreactor. In some embodiments, thebioreactor is a stirred suspension bioreactor. The method can furthercomprise isolating and purifying the variant cannabinoid synthase fromthe cell or the plurality of expanded cells. The cell can be a plantcell or a microorganism cell. The contacting can comprise delivering thenucleic acid construct or a vector comprising the nucleic acid constructinto the cell. The delivering can comprise microinjection,liposome-mediated transfection, electroporation, or nucleofection of thenucleic acid construct or vector comprising the nucleic acid constructinto the microorganism. In some embodiments, the method comprisesintegrating the nucleic acid construct encoding the variant cannabinoidsynthase into the genome of the cell.

Described herein, in certain embodiments, are methods of producing acannabinoid, comprising: (i) contacting a cell with a nucleic acidconstruct encoding the variant cannabinoid synthase, (ii) expressing thevariant cannabinoid synthase, and (iii) isolating a cannabinoid producedby the cell. In some embodiments, the method comprises expanding thecell to produce a plurality of expanded cells. In some embodiments, theexpanding occurs in a bioreactor. In some embodiments, the bioreactor isa stirred suspension bioreactor. The method can further compriseisolating and purifying the cannabinoid from the cell or the pluralityof expanded cells. The cell can be a plant cell or a microorganism cell.The contacting can occur in vivo. The contacting can occur ex vivo.Thecontacting can comprise delivering the nucleic acid construct or avector comprising the nucleic acid construct into the cell. Thedelivering can comprise microinjection, liposome-mediated transfection,electroporation, or nucleofection of the nucleic acid construct orvector comprising the nucleic acid construct into the microorganism. Insome embodiments, the method comprises integrating the nucleic acidconstruct encoding the variant cannabinoid synthase into the genome ofthe cell. Described herein, in certain embodiments, are methods ofproducing a cannabinoid, comprising: introducing a variant cannabinoidsynthase described herein to a substrate of the variant cannabinoidsynthase. In some embodiments, the substrate is a non-naturallyoccurring substrate described herein.

In some embodiments, libraries of mutations cannabinoid synthases areprovided, and screened to identify un-natural novel protein variantcannabinoid synthases that broaden the ranges of these basic functionalaspects of cannabinoid synthase and allow control of cannabinoidsynthesis that can produce specific small molecules at desired ratios(e.g., the desired ratio of CBDA to THCA, and the like). Expressing asoluble variant cannabinoid synthases that performs the same biochemicaltask as the native cannabinoid synthase, is useful in expressing thephytocannabinoid biosynthesis pathway exogenously in a microorganism.This permits for the expression of the most desirable cannabinoids,including non-abundant phytocannabinoids, at an industrial scale and inpure form to enable development of these phytocannabinoids into humantherapeutics.

In one embodiment, exemplary variant cannabinoid synthases have theamino acid sequences derived by translation of SEQ IDNO:DNA0001-DNA0678, corresponding to SEQ ID NO:1 through SEQ ID NO: 678,and combining any mutation found, but not limited to those set forth, inSEQ ID NO: DNA0001-DNA0678. As used herein, “protein variant” refers toan open reading frame with substitutions of amino acid residues relativeto WT. Examples of protein variants include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another; or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. Other illustrative examplesof protein variant substitutions include the changes of: alanine toserine; arginine to lysine; asparagine to glutamine or histidine;aspartate to glutamate; cysteine to serine; glutamine to asparagine;glutamate to aspartate; glycine to proline; histidine to asparagine orglutamine; isoleucine to leucine or valine; leucine to valine orisoleucine; lysine to arginine, glutamine, or glutamate; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; valine to isoleucine orleucine, and the like. The term “protein variant” also includes the useof a substituted amino acid in place of an unsubstituted amino acid.

Modifications and substitutions contemplated herein are not limited toreplacement of amino acids. For a variety of purposes, such as increasedstability, solubility, or configuration concerns, one skilled in the artwill recognize the need to introduce other modifications (e.g., bydeletion, replacement, or addition). Examples of such othermodifications include incorporation of rare amino acids, dextra-aminoacids, glycosylation sites, cytosine for specific disulfide bridgeformation. The modified peptides can be chemically synthesized, or theisolated gene can be site-directed mutagenized, or a synthetic gene canbe synthesized and expressed in bacteria, yeast, microalgae,baculovirus, any micro-organism or tissue culture, and the like.

Novel cannabinoid synthases that have less than 99% sequence identitywith the native consensus amino acid sequence (set forth in SEQ IDAA_consensus) are un-natural cannabinoid synthases, also referred toherein as non-naturally occurring cannabinoid synthases. Sequencehomology and identity are often measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). The term “non-identity” in thecontext of two or more nucleic acids or polypeptide sequences, refers totwo or more sequences or subsequences that have a specified percentageof amino acid residues or nucleotides that are not the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. The term“non-homology” in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are non-homologous or have a specified percentage of amino acidresidues or nucleotides that are non-homologous when compared andaligned for maximum correspondence over a comparison window ordesignated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. Programsreferred to hereinabove allow for substitution of an amino acid with asimilar amino acid by determining a degree of homology between thesequences being compared.

Lengthy table referenced here US20210238561A1-20210805-T00001 Pleaserefer to the end of the specification for access instructions.

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Lengthy table referenced here US20210238561A1-20210805-T00015 Pleaserefer to the end of the specification for access instructions.

EXAMPLES Materials and Methods

Library Construction: Libraries are constructed of novel variants ofCBDAS, THCAS and CBCAS based on novel amino acid substitutions and newcombinations of natural amino acid substitutions. These novel variantsare screened and investigated for altered enzyme repertoire. THCASdiverges from CBDAS, for example, at n=83 amino acid positions (FIG. 2).A rational enzyme engineering approach was employed to identify aminoacid positions with putative functional effects on synthase activityusing homology-based protein structure modeling for CBDAS and CBCAScompared to the reported THCAS crystal structure (Protein Data Bank IDcode 3VTE) in addition to comparative evolutionary analysis. In anotherembodiment, an approach to investigate all divergent sites for allpossible amino acids can be employed. This would result in a librarymatrix of size of 1660 variants (83 sites×20 amino acids=1660).

The following are the libraries designed in the invention:

Example 1

CBDAS site-saturation amino acid substitutions library: This library wascreated to identify a significantly improved CBDAS. The improved CBDASenzyme exhibits improved enzyme kinetics, and/or improved/alteredproduct profile and/or novel acceptance of substrate/s not accepted bythe consensus native synthase. Based on this homology-based proteinmodeling, the active site of CBDAS was shown to exhibit four amino aciddifferences from THCAS in the “deep active site” near the catalytic FADcofactor. [Note: A multiple alignment of the complete amino acidsequences between CBDAS and THCAS displayed an insertion/deletion eventat alignment position 253. Accordingly, THCAS displays a Serine (S)insertion at alignment site 253 (see FIG. 2). It follows that allhomologous amino acid positions>=253 through 544 on CBDAS, respectivelycorrelated to the homologous positions>=254 through 545 on THCAS]. Theidentified divergent amino acids in the “deep active site” were asfollows (according to standard IUPAC notation, the CBDAS residue islisted first, the CBDAS amino acid position is listed second, while thehomologous THCAS residue is listed last): H69Q, C180G, A414V (homologousto amino acid position 415 in THCAS), and I445T (homologous to aminoacid position 446 in THCAS). Four additional changes were alsoidentified in the “outer pocket” of CBDAS relative to THCAS. Thepositions of these substitutions on CBDAS are M256I, R295K, Q376K, andN377K. Variant cannabinoid synthases with amino acid substitutions atthese 8 sites were produced and assayed to explore the effect of aminoacid diversity on synthase function. Accordingly, each of the 8positions was substituted with the other 19 amino acids. First, (8sites×20 amino acids) n=160 deoxyribonucleic acid (DNA) sequences aregenerated based on a WT consensus DNA sequence of the open reading framefor native CBDAS (CBDAS DNA_consensus; SEQ ID NO: 1049; FIG. 6)including DNA mutations that translate into a single novel amino acidsubstitution for each protein variant. These n=160 CBDAS variants werelabeled SEQ ID NO: 1-SEQ ID NO: 160, and are described in Table 1. Aftersignal peptide cleavage these variants express as a ˜60 kDa soluble,monomeric protein with 522 residues (including 6 residues for HIS-taglabeling) and were purified by metal affinity chromatography. To assessenzyme activity of these CBDAS variants, the purified recombinantprotein was incubated with CBGA, and reaction products were analyzed byHPLC to determine whether the respective CBDAS variants show adifference in efficiency and/or kinetics of oxidocyclization conversionof CBGA and/or a change in the oxidocyclization product profile (i.e. %CBDA vs. % THCA or production of other cannabinoids). CBDAS variantshaving a difference in efficiency and/or kinetics of oxidocyclizationconversion of CBGA and/or a change in the oxidocyclization productprofile (i.e. % CBDA vs. % THCA or production of other cannabinoids)were isolated and provided as CBDAS variants.

Variant CBDA Synthase HPLC Assay

HPLC data was generated for a subset of CBDAS variants from Table 1described in Example 1. Experimental results demonstrated that novelCBDAS variants were generated demonstrating altered oxidocyclizationproduct profiles (FIG. 10). Specifically, select new variants were shownto produce varying proportions of CBDA and THCA compared to WT. While WTCBDA produced a majority of CBDA and a minority of THCA (6.8%; FIG. 9),some new CBDAS variants provided herein produced significantly more THCA(9.8%-71.8%) and some variants produce minimal amounts of THCA (FIG. 10and Table 7; Table 8 and FIGS. 11-14). Therefore, it was demonstratedthat rationally designed amino acid substitutions in a cannabinoidsynthase can significantly alter the oxidocyclization product profilesand proportion of cannabinoids produced. These novel cannabinoidsynthase enzymes were contemplated herein to be used for improvedcannabinoid synthase performance in biopharmaceutical or agriculturalapplications. Specifically, current data demonstrated that substitutionsat CBDAS amino acid positions 180, 414 and 445 have significant effectto increase THCA production. Alternatively, other substitutions at thesesame amino acid positions can significantly decrease THCA production byCBDAS variants. Thus, those of skill in the art are able to combinemultiple preferred amino substitutions provided herein to create alteredproduct profile and improved enzyme efficiency, as desired. Suchcombined variants will allow the creation of novel and superiorcannabinoid synthase enzymes for biopharmaceutical or agriculturalapplications.

Example 2

THCAS site-saturation amino acid substitutions library: This library iscreated to identify a significantly improved THCAS. The improved THCASenzyme exhibits improved enzyme kinetics, and/or improved/alteredproduct profile and/or novel acceptance of substrate/s not accepted bythe consensus synthase. This second library is constructed using thesame methods as described above for CBDAS to explore diversity in THCASproduct profile. We have identified n=8 amino acid positions in THCAS:(i.e. Q69, G180, V415, T446, 1257, K296, K377, and K378) for us inproducing variant THCA synthases. A library is created to determine theeffect of amino acid diversity by replacing at each of the 8 positions,the other 19 amino acids. First, (8 sites×20 amino acids) n=160 (8×20)160 deoxyribonucleic acid (DNA) sequences are generated based on a WTconsensus DNA sequence of the open reading frame for native THCAS (THCASDNA_consensus; SEQ ID NO: 1050; FIG. 7) including DNA mutations thattranslate into a single novel amino acid substitution for each proteinvariant. These n=160 THCAS variants are labeled SEQ ID NO:161-SEQ ID NO:320 (Table 2). After signal peptide cleavage these variants express as a˜60 kDa soluble, monomeric protein with 520 residues (including 6residues for HIS-tag labeling) and are purified by metal affinitychromatography. To assess enzyme activity of these THCAS variants, thepurified recombinant protein is incubated with CBGA and reactionproducts are analyzed by HPLC to determine whether the respective THCASvariants demonstrate a difference in efficiency and/or kinetics ofoxidocyclization conversion of CBGA and/or a change in theoxidocyclization product profile (i.e. % CBDA vs. % THCA or productionof other cannabinoids, such as CBCA). THCAS variants having a differencein efficiency and/or kinetics of oxidocyclization conversion of CBGAand/or a change in the oxidocyclization product profile (i.e. % CBDA vs.% THCA or production of other cannabinoids, such as CBCA) are isolatedand provided as THCAS variants.

Example 3

THCAS combinatorial amino acid substitution library to convert THCAS toa CBDAS: This library was created to identify a significantly improvedCBDAS by converting a native THCAS into a CBDAS. The improved CBDASenzyme exhibits improved enzyme kinetics, and/or improved/alteredproduct profile. The third library was constructed by specificsubstitutions at the deep active site amino acid positions to convertTHCAS to a novel and improved synthase that produces CBDA. The 4 aminoacid positions in the deep active site were mutated to the correspondingCBDAS amino acid. These substitutions were Q69H, G180C, V415A, andT446I. These variants consisted of all possible combinations of single,double, triple, and quadruple substitutions resulting in 15 THCASvariants corresponding to SEQ ID NO: SEQ ID NO: 321 to SEQ ID NO: 335,set forth in Table 3. To assess enzyme activity of these THCAS variants,the purified recombinant protein were incubated with CBGA and reactionproducts were analyzed by HPLC to determine whether the respective THCASvariants showed a difference in efficiency and/or kinetics ofoxidocyclization conversion of CBGA and/or a change in theoxidocyclization product profile (% CBDA or % CBDA vs. % THCA orproduction of other cannabinoids). THCAS variants having a difference inefficiency and/or kinetics of oxidocyclization conversion of CBGA and/ora change in the oxidocyclization product profile (i.e. % CBDA vs. % THCAor production of other cannabinoids, such as CBCA) were isolated andprovided as THCAS variants.

Variant THCA Synthase HPLC Assay

HPLC data was generated as set forth above in Example 3 for all THCAsynthase variants from Table 3. The experimental results demonstratedthat THCAS variants were created with altered oxidocyclization productprofiles. Specifically, select new variants were shown to producesignificantly increased proportions of CBDAS (up to 8.59% CBDAS), whileWT THCAS produced only a negligible proportion of CBDAS (0.0%-0.25%CBDAS) (FIG. 8; Table 6). In addition to the increased production ofCBDAS, some select variants exhibited novel production of CBCAS (albeitin fairly low amounts), while WT THCAS did not produce any detectableamount of CBCAS (FIG. 9; Table 7). Comparison of the four singletonmutation variants indicated that enzymes derived from SEQ ID NO: 323(V415A) and SEQ ID NO: 324 (T446I) produced a higher proportion of CBDAScompared to enzymes derived from SEQ ID NO: 321 (Q69H) and SEQ ID NO:322 (G180C). The underlying mutations at V415A and T446I were preferredsubstitutions and these amino acid position sites were rationallyidentified candidates for site saturation mutagenesis library screening.Variant cannabinoid synthases (e.g., THCA synthases or CBDA synthases)that included unnaturally occurring substitutions at these two sites(V415 and T446) were provided herein as improved cannabinoid synthases.From the results obtained herein shown in FIG. 8, variant SEQ ID NO:330, it was contemplated that stacking these two preferred amino acidsubstitutions would have a synergistic effect to increase the proportionof CBDAS production to a proportion that is greater than eithermutation's effect independently (see, e.g., FIG. 8, variant SEQ ID NO:330). Accordingly, provided herein are novel cannabinoid synthase (e.g.,CBDAS, THCAS or CBCAS) variants that include combinations of two or moresubstitutions across sites 415 and 446. Furthermore, these samepreferred amino acid substitutions (i.e. V415A and T446I) appeared tocause de novo production of CBCA (while WT THCAS does not produce anydetectible amount of CBCAS) (FIG. 9). It was contemplated herein todetermine the effect of different amino acid substitutions at these twosites (i.e. V415A and T446I) for altered oxidocyclization productprofiles specifically related to both CBDA and CBCA.

Example 4

CBDAS combinatorial amino acid substitution library using naturalsubstitution of native CBDAS: This library is created to identify asignificantly improved CBDAS. The improved variant CBDAS enzyme exhibitsimproved enzyme kinetics, and/or improved/altered product profile and/ornovel acceptance of substrate/s not accepted by the consensus CBDASsynthase. The fourth library is constructed to assay novel combinationsof natural substitutions of native CBDAS. Theses substitutions are notpresent in SEQ ID NO: CBDAS_E551071 (SEQ ID NO: 1043), yet are found innature on other functional alleles of CBDAS. It is contemplated that anoptimal combination of natural substitutions provides a novel CBDASvariant not found in nature with an improved enzyme functionalrepertoire, e.g., showing a difference in efficiency and/or kinetics ofoxidocyclization conversion of CBGA and/or a change in theoxidocyclization product profile (% CBDA or % CBDA vs. % THCA orproduction of other cannabinoids). CBDAS variants having a difference inefficiency and/or kinetics of oxidocyclization conversion of CBGA and/ora change in the oxidocyclization product profile (i.e. % CBDA vs. % THCAor production of other cannabinoids, such as CBCA) are isolated andprovided as CBDAS variants. In another embodiment, the variant aminoacids in variant CBDAS having natural amino acid substitutions arecombined with novel substitutions found in other libraries describedherein to further improve product specificity. It is contemplated hereinthat such a combined enzyme with multiple substitutions is asignificantly improved novel variant enzyme optimized for industrialproduction of cannabinoids. Sequence heterogeneity amongst CBDASsequences from multiple strains of “drug type” and “fiber type” plantsrevealed polymorphisms among CBDAS sequences. Six (n=6) amino acidpositions in CBDAS were identified as polymorphic sites that retained afunctional CBDAS. The natural substitutions identified at thesepositions were T74S, N168S, N196S, K474Q, R543H, and H143R. A library ofvariants is constructed with all possible single, double, triple,quadruple, quintuple, and sextuple combinations (respectively,n=6+15+20+15+6+1=63). These variants are created on the sequencebackbone of CBDAS_DNA_consensus (SEQ ID NO: 1046; correlating to aminoacid CBDAS_E551071; FIG. 2). This yields n=63 CBDAS variants SEQ ID NO:336 to SEQ ID NO: 398 (Table 4). To assess enzyme activity of theseCBDAS variants, the purified recombinant protein are incubated with CBGAand reaction products are analyzed by HPLC to determine whether therespective CBDAS variants show a difference in efficiency and/orkinetics of oxidocyclization conversion of CBGA and/or a change in theoxidocyclization product profile (% CBDA vs. % THCA or production ofother cannabinoids). CBDAS variants having a difference in efficiencyand/or kinetics of oxidocyclization conversion of CBGA and/or a changein the oxidocyclization product profile (i.e. % CBDA vs. % THCA orproduction of other cannabinoids) are isolated and provided as CBDASvariants.

Example 5

CBCAS site saturation amino acid substitution library: This library iscreated to identify a significantly improved CBCAS. The variant CBCASenzyme exhibits improved enzyme kinetics, and/or improved/alteredproduct profile and/or novel acceptance of substrate/s not accepted bythe consensus synthase. The fifth library is constructed based onputatively functional amino acid sites (n=14) in CBCAS that have beenidentified by combined comparative evolutionary analysis andhomology-based modeling. Each site is altered to all remaining naturalalternative amino acid substitutions (n=19) using as a template for theconsensus natural sequence the SEQ ID NO: CBCAS_JP2016 (FIG. 2)_JP2016(SEQ ID NO: 1045). This results in screening of 20 variants per site.The resultant n=280 DNA sequences are presented in Table 5 (SEQ ID NO:399 to SEQ ID NO: 678)). To assess enzyme activity of these CBCASvariants, the purified recombinant protein are incubated with CBGA andreaction products are analyzed by HPLC to determine whether any of therespective CBCAS variants show an altered oxidocyclization conversion ofCBGA to CBCA and/or a change in the oxidocyclization product profile(for example % THCAS vs. % CBCAS or production of other natural or novelcannabinoids). CBCAS variants having an altered oxidocyclizationconversion of CBGA to CBCA and/or a change in the oxidocyclizationproduct profile (for example % THCAS vs. % CBCAS or production of othernatural or novel cannabinoids) are isolated and provided herein.

Example 6

The cannabinoid synthase variants are studied as follows:

A) Construction of a synthesized gene library of (A) n=160 CBDASvariants with select amino acid substitutions as described in EXAMPLE 1(B) n=160 THCAS variants with select amino acid substitutions asdescribed in EXAMPLE 2, (C) n=15 THCAS convert into CBDAS variants asdescribed in EXAMPLE 3, (D) the (63) novel natural combinatorialvariants of CBDAS based on natural amino acid substitutions as describedin EXAMPLE 4 and (E) n=28 CBCAS variants with site saturation at 14select sites as described in EXAMPLE 5.

DNA primers are designed, ordered and synthesized, that allowsite-directed mutagenesis in a Saccharomyces cerevisiae yeast proteinexpression plasmid encoding the native cannabinoid synthases(THCAS_DNA_consensus (SEQ ID NO: 1047), CBDAS_DNA_consensus (SEQ ID NO:1046), and CBCAS_DNA_consensus (SEQ ID NO:1048)). Agilent “Quik ChangeII Site-Directed Mutagenesis Kit (#2000523) is used to create variantsof cannabinoid synthases. The sequences for the cannabinoid synthasesvariants are set forth in the Tables herein as SEQ ID NO: 1 to SEQ IDNO:678 (which are also referred to herein as DNA_0001 to DNA_0678). Eachcannabinoid synthase variant contains a unique single amino acidsubstitution or multiple amino acid substitutions relative to the basenatural consensus sequence constructs. This is done for n=160 CBDASvariants with select amino acid substitutions, (B) n=160 THCAS variantswith select amino acid substitutions, (C) 15 THCAS variants withmultiple substitutions to create a CBDAS variant and (D) n=63) CBDASvariants which are novel combinations of natural substitutions and (E)n=280 CBCAS variants with site saturation at select sites to create animproved CBCAS variant.

Each of these variant synthases demonstrating an altered synthasefunctional repertoire is provided herein as a variant cannabinoidsynthase (e.g., a variant CBDAS, THCAS or CBCAS).

The variant sequences are described and set forth herein as SEQ ID NO: 1to SEQ ID NO: 678. Each cannabinoid synthase variant contains a uniqueamino acid or multiple amino acid substitutions relative to the basenative constructs (SEQ ID NO:1046 to SEQ ID NO: 1048).

B) Expression and Purification of Proteins from the SynthesizedCannabinoid Synthase Variants Libraries.

DNA plasmids containing each of the cannabinoid synthase variants areindividually transformed into S. cerevisiae yeast stain YPH857 by usingchemically competent YPH857 yeast cells created by lithium acetatetransformation protocol in the Yeast Protocols Handbook provided byClonetech Laboratories (www. clontech.com). This produces individual Scerevisiae yeast stains, each containing a yeast expression plasmidencoding a single cannabinoid synthase variant.

To induce protein expression, individual yeast strains encoding each ofthe “cannabinoid synthase variants” driven by a yeast constitutivepromoter, are individually inoculated into 100 milliliters of selectminimal yeast media with in a 250 milliliter culture flask and grown at30 degrees Celsius until saturation (˜3-4 days) with vigorous shaking.Upon reaching saturation, each culture is diluted into 1000 millilitersof YPDA yeast media in a 2000 milliliter culture flask and grown at 30degrees Celsius for 24 hours with vigorous shaking. After 24 hours eachyeast culture is harvested by spinning down at 4,000 G and thesupernatant was removed and the yeast pellets were saved for targetprotein extraction.

Each individual yeast cell pellets is resuspended in 25 milliliters of asolution containing 50 millimolar Tris-HCL, 500 millimolar sodiumchloride, 5 millimolar imidazole, and 10% glycerol pH 7.8 (“lysisbuffer”), resulting in a “cell slurry.” To each individual “cellslurry”, 30 microliters of 25 units per microliter Benzonase (Millipore,Benzonase, catalog number 70664-1), as well as 300 microliters ofphosphatase and protease inhibitor (Thermo-Fisher, Halt Protease andPhosphatase Inhibitor Cocktail, EDTA-free, catalog number 78441) and 10mg of Zymolyase®-20T (Sunrise Science Products, catalog number N0766391)are added. Each “cell slurry” is incubated with gentle agitation for 30minutes at 370 C. After incubation each individual “cell slurry” is thensubjected to 30 second pulses of sonication, 4 times each, for a totalof 120 seconds, using the Fisher Scientific Sonic Dismembrator Model 500under 30% amplitude conditions. In between each 30 second pulse ofsonication, the “cell slurry” is placed on ice for 30 seconds. Aftersonication, each “cell slurry” is centrifuged for 45 minutes at 14,000times gravity to separate the soluble and insoluble fractions.

Protein purification columns (Bio-Rad, Econo-Pac Chromotography Columns,catalog number 7321010) are prepared by adding 1.5 milliliters TALON®Superflow Metal Affinity Resin slurry (Takara, TALON® Superflow MetalAffinity Resin, catalog number 635506). 5 milliliters deionized water isadded to resin slurry, to agitate and rinse the resin. The columns arethen uncapped and the resulting flow-through was discarded. Then, 5milliliters deionized water are added a second time, and the resultingflow-through is discarded. Then, 10 milliliters “lysis buffer” is addedto the resin, completely disturbing the resin bed, and the flow-throughwill be discarded.

The protein purification columns are capped, and the supernatant fromthe “cell slurry” is added to the resin bed without disturbing the resinbed. The columns are uncapped, allowing the supernatant to pass over theresin bed. The resin is then washed 2 times with 10 milliliters of asolution containing 50 millimolar Tris-HCl, 500 millimolar sodiumchloride, and 20 millimolar imidazole pH 7.8 (“wash buffer”). Theflow-through from the wash steps is discarded. The protein is theneluted off the column with 7.5 milliliters of a solution containing 50millimolar Tris-HCl, 200 millimolar sodium chloride, and 250 millimolarimidazole pH 7.8. The eluted protein is collected and dialyzed overnightin 4 liters of a solution containing 100 millimolar sodium citrate pH5.5 in 3.5-5.0 kilodalton dialysis tubing (Spectrum Labs, Spectra/Pordialysis tubing, catalog number 133198). After overnight dialysis,protein is concentrated to approximately 100 uL using centrifugalprotein filters (Millipore Amicon Ultra-15 Ultracel 10K, catalog numberUFC901024). UV absorbance at 280 nm is used for measurement ofconcentrated proteins to estimate cannabinoid synthase variant yield perliter of yeast culture.

C) Screening of the Cannabinoid Synthase Protein Variants for ProteinActivity and Phenotypes.

The library of cannabinoid synthase variants is screened for proteinexpression by western blot with an anti-HIS antibody (Cell SignalingTechnologies, anti-his monoclonal antibody, catalog number 23655)according to the protocol provided by Cell Signaling Technologies forthe antibody. Any variants that demonstrate detectable levels of proteinexpression as determined by western blot are used in an oxidocyclizationassay with CBGA.

Proteins that exhibited detectable expression by western blot areassayed for oxidocyclization activity using CBGA as a substrate. Eachreaction is performed in a volume of 20-100 microliters and contained100 millimolar Sodium Citrate pH 5.5, 0.2 millimolar CBGA and activecannabinoid synthase variant protein. These reactions are incubated for16 hours at 37° C.

To assess oxidocyclization activity by HPLC the oxidocyclizationproducts are extracted from the assay reaction with the followingprotocol: 2:1 ratio v/v of ethyl acetate:reaction mix is added to eachreaction and vortexed thoroughly. After vortexing, each reaction iscentrifuged for 1 minute at 14,000 G. The top layer (“organic layer”) iscollected. This is repeated twice. The collected organic layer isevaporated, and the resulting residue is resuspended in 40 microlitersof 100% methanol. After resuspending in methanol, 40 microliters of 100%HPLC grade water is added to bring the final solution to 50% methanol.These are referred to as the “variant reactions with CBGA.”

The final 50% methanol solutions are run on a Thermo Fisher UltiMate3000 UHPLC with an Acclaim RSLC 120 angstrom C18 column with a 4millimeter Phenomonex Securityguard guard column (54 millimeter totalcolumn length). Product is detected by ultraviolet light absorption at270 nm.

D) Assigning Individual Cannabinoid Synthase Variants to SpecificProtein Activity and Phenotypes. Example 7

CBDAS stacking of mutations: Based on the data obtained from the CBDAsite-saturation amino acid substitution library (Table 8), 7 mutationswere chosen for a subsequent analysis of double and triple mutationcombinations based on their ability to result in an increased yield ofcannabinoids relative to a wild type CBDAS or an increased proportion ofCBDA:THCA relative to a wild type CBDAS. These mutations included H69K,H69Q, H69V, H69G, A414V, A414I, and I445M.

Double mutations examined included: H69K/A414V, H69Q/A414V, H69V/A414V,H69G/A414V, H69K/A414I, H69Q/A414I, H69V/A414I, H69G/A414I, A414V/1445M,A414I/1445M, H69K/1445M, H69Q/I445M, H69V/I445M, and H69G/I445M. Triplemutations examined included: H69K/A414V/1445M, H69Q/A414V/1445M,H69V/A414V/1445M, H69G/A414V/1445M, H69K/A414I/1445M, H69Q/A414I/1445M,H69V/A414I/1445M, and H69G/A414I/445M. Results are displayed in Table11, Table 12, and FIGS. 15-17.

The above description of the disclosed embodiments is provided to enableany person skilled in the art to make or use the described compositionsand perform the described methods. Various modifications to theseembodiments will be readily apparent to those skilled in the art, andthe generic principles described herein can be applied to otherembodiments without departing from the spirit or scope of thedisclosure. Thus, it is to be understood that the description anddrawings presented herein represent a presently preferred embodiment andare therefore representative of the subject matter which is broadlycontemplated by the present disclosure. It is further understood thatthe scope of the present disclosure fully encompasses other embodimentsthat may become obvious to those skilled in the art and that the scopeof the present disclosure is accordingly not limited.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210238561A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A variant cannabinoid synthase or an activefragment thereof comprising a non-naturally occurring amino acidsequence relative to a wild-type cannabinoid synthase or an activefragment thereof which acts on a substrate to produce an altered amountof a cannabinoid relative to an amount of the cannabinoid produced bythe wild-type cannabinoid synthase or active fragment thereof.
 2. Thevariant cannabinoid synthase or active fragment thereof of claim 1,wherein the variant cannabinoid synthase is a variant cannabidiolic acid(CBDA) synthase comprising a non-naturally occurring amino acid sequencerelative to a wild type CBDA synthase of SEQ ID NO:
 1043. 3. The variantcannabinoid synthase or active fragment thereof of claim 1, wherein thevariant cannabinoid synthase is a variant cannabidiolic acid (CBDA)synthase comprising a non-naturally occurring amino acid sequencerelative to a wild type consensus CBDA synthase of SEQ ID NO:
 1046. 4.The variant cannabinoid synthase or active fragment thereof of claim 1,wherein the variant cannabinoid synthase is a tetrahydrocannabinolicacid (THCA) synthase comprising a non-naturally occurring amino acidsequence relative to a wild type THCA synthase of SEQ ID NO:
 1044. 5.The variant cannabinoid synthase or active fragment thereof of claim 1,wherein the variant cannabinoid synthase is a tetrahydrocannabinolicacid (THCA) synthase comprising a non-naturally occurring amino acidsequence relative to a wild type consensus THCA synthase of SEQ ID NO:1047.
 6. The variant cannabinoid synthase or active fragment thereof ofclaim 1, wherein the variant cannabinoid synthase is a cannabichromenicacid (CBCA) synthase comprising a non-naturally occurring amino acidsequence relative to a wild type CBCA synthase of SEQ ID NO:
 1045. 7.The variant cannabinoid synthase or active fragment thereof of claim 1,wherein the variant cannabinoid synthase is a cannabichromenic acid(CBCA) synthase comprising a non-naturally occurring amino acid sequencerelative to a wild type consensus CBCA synthase of SEQ ID NO:
 1048. 8.The variant cannabinoid synthase or active fragment thereof of claim 1,wherein the cannabinoid is selected from the group consisting of:tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC),tetrahydrocannabinvarin (THCV), cannabidiolic acid (CBDA), cannabidiol(CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabichromevarin(CBCV), cannabichromenic acid (CBCA), and a combination thereof.
 9. Thevariant cannabinoid synthase or active fragment thereof of claim 1,wherein the substrate is a naturally occurring substrate.
 10. Thevariant cannabinoid synthase or active fragment thereof of claim 9,wherein the naturally occurring substrate is selected from the groupconsisting of cannabigerol (CBG), cannabigerolic acid (CBGA),cannbigerovarinic acid (GBGVA), and any homolog thereof.
 11. The variantcannabinoid synthase or active fragment thereof of claim 1, wherein thesubstrate is a non-naturally occurring substrate.
 12. The variantcannabinoid synthase or active fragment thereof of claim 11, wherein thenon-naturally occurring substrate can comprise: a non-naturallyoccurring tail variant, a prenyl donor, or a combination thereof. 13.The variant cannabinoid synthase or active fragment thereof of claim 1,wherein the altered amount of the cannabinoid produces a change in aproportion of a first cannabinoid to a second cannabinoid.
 14. Thevariant cannabinoid synthase or active fragment thereof of claim 13,wherein the variant cannabinoid synthase is a variant CBDA synthase. 15.The variant cannabinoid synthase or active fragment thereof of claim 14,wherein the first cannabinoid is CBDA and the second cannabinoid isTHCA.
 16. The variant cannabinoid synthase or active fragment thereof ofclaim 15, wherein a proportion of CBDA:THCA produced by a wild type CBDAsynthase is about 95:5.
 17. The variant cannabinoid synthase or activefragment thereof of claim 14, wherein the non-naturally occurring aminoacid sequence comprises an amino acid mutation at position 445 relativeto a wild-type consensus CBDA synthase amino acid sequence set forth inSEQ ID NO:
 1045. 18. The variant cannabinoid synthase or active fragmentthereof of claim 17, wherein the amino acid mutation at position 445 isselected from the group consisting of: I445M and I445L.
 19. The variantcannabinoid synthase or active fragment thereof of claim 1, wherein thealtered amount of the cannabinoid is an increase or a decrease in ayield of the cannabinoid.
 20. The variant cannabinoid synthase or activefragment thereof of claim 19, wherein the yield is a nMol of thecannabinoid produced per milligram of the substrate.
 21. The variantcannabinoid synthase or active fragment thereof of claim 19, wherein thedecrease in the yield is less than 100%, less than 200%, or less than300% of a yield of the cannabinoid of the wild-type cannabinoid synthaseor active fragment thereof.
 22. The variant cannabinoid synthase oractive fragment thereof of claim 19, wherein the increase in the yieldis more than 100%, more than 200%, or more than 300% of a yield of thecannabinoid of the wild-type cannabinoid synthase or active fragmentthereof.
 23. The variant cannabinoid synthase or active fragment thereofof claim 22, wherein the variant cannabinoid synthase is a variant CBDAsynthase.
 24. The variant cannabinoid synthase or active fragmentthereof of claim 23, wherein the non-naturally occurring amino acidsequence comprises at least one amino acid mutation at position 69, 414,or 445 relative to a wild-type consensus CBDA synthase amino acidsequence set forth in SEQ ID NO:
 1045. 25. The variant cannabinoidsynthase or active fragment thereof of claim 24, wherein the at leastone amino acid mutation at position 69 is selected from the groupconsisting of: H69R, H69G, H69K, H69Q, H69A, and H69V.
 26. The variantcannabinoid synthase or active fragment thereof of claim 24, wherein theat least one amino acid mutation at position 414 is selected from thegroup consisting of: A414T, A414I, and A414V.
 27. The variantcannabinoid synthase or active fragment thereof of claim 24, wherein theat least one amino acid mutation at position 445 is 1445V.
 28. Thevariant cannabinoid synthase or active fragment thereof of claim 23,wherein the non-naturally occurring amino acid sequence comprises atleast two amino acid mutations at positions selected from the groupconsisting of: 69, 180, 414, and 445 relative to a wild-type consensusCBDA synthase amino acid sequence set forth in SEQ ID NO:
 1045. 29. Thevariant cannabinoid synthase or active fragment thereof of claim 28,wherein one of the at least two amino acid mutations is at amino acidposition
 69. 30. The variant cannabinoid synthase or active fragmentthereof of claim 29, wherein one of the at least two amino acidmutations at amino acid position 69 is selected from the groupconsisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I, H69K, H69L,H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, and H69Y.
 31. Thevariant cannabinoid synthase or active fragment thereof of claim 29,wherein one of the at least two amino acid mutations at amino acidposition 69 is selected from the group consisting of H69K, H69Q, H69V,and H69G.
 32. The variant cannabinoid synthase or active fragmentthereof of claim 28, wherein one of the at least two amino acidmutations is at amino acid position
 180. 33. The variant CBDA synthaseor active fragment thereof of claim 32, wherein one of the at least twoamino acid mutations at amino acid position 180 is selected from thegroup consisting of C180A, C180D, C180E, C180F, C180G, C180H, C180I,C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V,C180W, and C180Y.
 34. The variant cannabinoid synthase or activefragment thereof of claim 28, wherein one of the at least two amino acidmutations is at amino acid position
 414. 35. The variant cannabinoidsynthase or active fragment thereof of claim 34, wherein one of the atleast two amino acid mutations at amino acid position 414 is selectedfrom the group consisting of A414C, A414D, A414E, A414F, A414G, A414H,A414I, A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T,A414V, A414W, and A414Y.
 36. The variant cannabinoid synthase or activefragment thereof of claim 34, wherein one of the at least two amino acidmutations at amino acid position 414 is selected from the groupconsisting of: A414V and A414I.
 37. The variant cannabinoid synthase oractive fragment thereof of claim 28, wherein one of the at least twoamino acid mutations is at amino acid position
 445. 38. The variantcannabinoid synthase or active fragment thereof of claim 37, wherein oneof the at least two amino acid mutations at amino acid position 445 isselected from the group consisting of I445A, I445C, I445D, I445E, I445F,I445G, I445H, 1445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S,I445T, I445V, 1445W, and I445Y.
 39. The variant cannabinoid synthase oractive fragment thereof of claim 37, wherein one of the at least twoamino acid mutations at amino acid position 445 is I445M.
 40. Thevariant cannabinoid synthase or active fragment thereof of claim 28,wherein the at least two amino acid mutations are at a pair of positionsselected from the group consisting of: 69/180, 69/414, 69/445, 180/414,180/445, and 414/445.
 41. The variant cannabinoid synthase or activefragment thereof of claim 28, wherein the at least two amino acidmutations are selected from the group consisting of: A414V/H69K,A414V/H69Q, A414V/H69V, A414V/H69G, A414V/I44M, A414I/H69K, I445M/H69K,and I445M/H69Q.
 42. The variant cannabinoid synthase or active fragmentthereof of claim 1, wherein the non-naturally occurring amino acidsequence comprises at least three amino acid mutations at positionsselected from the group consisting of: 69, 180, 414, and 445 relative toa wild-type consensus CBDA synthase amino acid sequence set forth in SEQID NO:
 1045. 43. The variant cannabinoid synthase or active fragmentthereof of claim 42, wherein the at least three amino acid mutations areat a triple of positions selected from the group consisting of:69/180/441, 69/180/445, 69/414/445, and 180/414/445.
 44. The variantcannabinoid synthase or active fragment thereof of claim 42, wherein theat least three amino acid mutations are H69Q/A414V/I445M.
 45. A variantcannabidiolic acid (CBDA) synthase or active fragment thereof comprisingan amino acid mutation at a position selected from the group consistingof: 69, 414, 180, and 445 relative to a wild-type consensus CBDAsynthase set forth in SEQ ID NO:
 1045. 46. The variant CBDA synthase oractive fragment thereof of claim 45, wherein a mutation at amino acidposition 69 is selected from the group consisting of: H69R, H69G, H69K,H69Q, H69A, and H69V.
 47. The variant CBDA synthase or active fragmentthereof of claim 46, wherein the mutation at amino acid position 69results in the variant CBDA synthase producing an increase yield of aCBDA relative to a wild type CBDA synthase or active fragment thereof.48. The variant CBDA synthase or active fragment thereof of claim 47,wherein the yield of the variant or active fragment thereof is more than100%, more than 200%, or more than 300% of a yield of CBDA of thewild-type CBDA synthase or active fragment thereof
 49. The variant CBDAsynthase or active fragment thereof of claim 48, wherein the yield is anMol of CBDA produced per milligram of a substrate.
 50. The variant CBDAsynthase or active fragment thereof of claim 49, wherein the substrateis cannabigerolic acid (CBGA).
 51. The variant CBDA synthase or activefragment thereof of claim 45, wherein a mutation at amino acid position414 is selected from the group consisting of: A414T, A414I, and A414V.52. The variant CBDA synthase or active fragment thereof of claim 51,wherein the mutation at amino acid position 414 results in the variantCBDA synthase producing an increase yield of a CBDA relative to a wildtype CBDA synthase or active fragment thereof.
 53. The variant CBDAsynthase or active fragment thereof of claim 52, wherein the yield ofthe variant or active fragment thereof is more than 100% or more than200% of a yield of CBDA of the wild-type CBDA synthase or activefragment thereof.
 54. The variant CBDA synthase or active fragmentthereof of claim 53, wherein the yield is a nMol of CBDA produced permilligram of a substrate.
 55. The variant CBDA synthase or activefragment thereof of claim 54, wherein the substrate is cannabigerolicacid (CBGA).
 56. The variant CBDA synthase or active fragment thereof ofclaim 45, wherein a mutation at amino acid position 445 is selected fromthe group consisting of: I445M, I445L, and 1445V.
 57. The variant CBDAsynthase or active fragment thereof of claim 56, wherein the mutation1445V results in the variant CBDA synthase producing an increase yieldof a CBDA relative to a wild type CBDA synthase or active fragmentthereof.
 58. The variant CBDA synthase or active fragment thereof ofclaim 57, wherein the yield of the variant or active fragment thereof ismore than 100% of a yield of CBDA of the wild-type CBDA synthase oractive fragment thereof
 59. The variant CBDA synthase or active fragmentthereof of claim 58, wherein the yield is a nMol of CBDA produced permilligram of a substrate.
 60. The variant CBDA synthase or activefragment thereof of claim 59, wherein the substrate is cannabigerolicacid (CBGA).
 61. The variant CBDA synthase or active fragment thereof ofclaim 56, wherein the mutation I445L or I445M results in the variantCBDA synthase produces an increased proportion of cannabidiolic acid(CBDA): tetrahydrocannabinolic acid (THCA) relative to a wild type CBDAsynthase or active fragment thereof
 62. The variant CBDA synthase oractive fragment thereof of claim 61, wherein a proportion of CBDA:THCAproduced by the wild type CBDA synthase or active fragment thereof isabout 95:5.
 63. A variant cannabidiolic acid (CBDA) synthase or activefragment thereof comprising at least two amino acid mutations at aminoacid positions selected from the group consisting of: 69, 180, 414, and445, relative to a wild-type consensus CBDA synthase set forth in SEQ IDNO:
 1045. 64. The variant CBDA synthase or active fragment thereof ofclaim 63, wherein the at least two amino acid mutations result in thevariant CBDA synthase producing an increase yield of a CBDA relative toa wild type CBDA synthase or active fragment thereof.
 65. The variantCBDA synthase or active fragment thereof of claim 64, wherein the yieldof the variant or active fragment thereof is more than 100%, more than200%, more than 300%, more than 400%, more than 500%, more than 600%,more than 700%, or more than 800% of a yield of CBDA of the wild-typeCBDA synthase or active fragment thereof
 66. The variant CBDA synthaseor active fragment thereof of claim 65, wherein the yield is a nMol ofCBDA produced per milligram of a substrate.
 67. The variant CBDAsynthase or active fragment thereof of claim 66, wherein the substrateis cannabigerolic acid (CBGA).
 68. The variant CBDA synthase or activefragment thereof of claim 63, wherein the variant CBDA synthase oractive fragment thereof produces an increased proportion ofcannabidiolic acid (CBDA): tetrahydrocannabinolic acid (THCA) relativeto the wild-type CBDA synthase or active fragment thereof
 69. Thevariant CBDA synthase or active fragment thereof of claim 68, wherein aproportion of CBDA:THCA produced by the wild type CBDA synthase oractive fragment thereof is about 95:5.
 70. The variant CBDA synthase oractive fragment thereof of claim 63, wherein one of the at least twoamino acid mutations is at amino acid position
 69. 71. The variant CBDAsynthase or active fragment thereof of claim 70, wherein one of the atleast two amino acid mutations at amino acid position 69 is selectedfrom the group consisting of H69A, H69C, H69D, H69E, H69F, H69G, H69I,H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V, H69W, andH69Y.
 72. The variant CBDA synthase or active fragment thereof of claim70, wherein one of the at least two amino acid mutations at amino acidposition 69 is selected from the group consisting of H69K, H69Q, H69V,and H69G.
 73. The variant CBDA synthase or active fragment thereof ofclaim 63, wherein one of the at least two amino acid mutations is atamino acid position
 180. 74. The variant CBDA synthase or activefragment thereof of claim 73, wherein one of the at least two amino acidmutations at amino acid position 180 is selected from the groupconsisting of C180A, C180D, C180E, C180F, C180G, C180H, C180I, C180K,C180L, C180M, C180N, C180P, C180Q, C180R, C180S, C180T, C180V, C180W,and C180Y.
 75. The variant CBDA synthase or active fragment thereof ofclaim 73, wherein one of the at least two amino acid mutations is atamino acid position
 414. 76. The variant CBDA synthase or activefragment thereof of claim 75, wherein one of the at least two amino acidmutations at amino acid position 414 is selected from the groupconsisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I, A414K,A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V, A414W,and A414Y.
 77. The variant CBDA synthase or active fragment thereof ofclaim 75, wherein one of the at least two amino acid mutations at aminoacid position 414 is selected from the group consisting of: A414V andA414I.
 78. The variant CBDA synthase or active fragment thereof of claim63, wherein one of the at least two amino acid mutations is at aminoacid position
 445. 79. The variant CBDA synthase or active fragmentthereof of claim 78, wherein one of the at least two amino acidmutations at amino acid position 445 is selected from the groupconsisting of I445A, I445C, I445D, 1445E, I445F, I445G, I445H, 1445K,I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T, I445V, 1445W,and I445Y.
 80. The variant CBDA synthase or active fragment thereof ofclaim 78, wherein one of the at least two amino acid mutations at aminoacid position 445 is I445M.
 81. The variant CBDA synthase or activefragment thereof of claim 63, wherein the at least two amino acidmutations are selected from the group consisting of: A414V/H69K,A414V/H69Q, A414V/H69V, A414V/H69G, A414V/I44M, A414I/H69K, I445M/H69K,and I445M/H69Q.
 82. The variant CBDA synthase or active fragment thereofof claim 63, wherein the variant CBDA synthase comprises at least threeamino acid mutations at amino acid positions selected from the groupconsisting of: 69, 180, 414, and 445, relative to a wild-type consensusCBDA synthase set forth in SEQ ID NO:
 1045. 83. The variant CBDAsynthase or active fragment thereof of claim 82, wherein one of the atleast three amino acid mutations is at amino acid position
 69. 84. Thevariant CBDA synthase or active fragment thereof of claim 83, whereinone of the at least three amino acid mutations at amino acid position 69is selected from the group consisting of H69A, H69C, H69D, H69E, H69F,H69G, H69I, H69K, H69L, H69M, H69N, H69P, H69Q, H69R, H69S, H69T, H69V,H69W, and H69Y.
 85. The variant CBDA synthase or active fragment thereofof claim 83, wherein one of the at least three amino acid mutations atamino acid position 69 is selected from the group consisting of H69K,H69Q, H69V, and H69G.
 86. The variant CBDA synthase or active fragmentthereof of claim 82, wherein one of the at least three amino acidmutations is at amino acid position
 180. 87. The variant CBDA synthaseor active fragment thereof of claim 86, wherein one of the at leastthree amino acid mutations at amino acid position 180 is selected fromthe group consisting of C180A, C180D, C180E, C180F, C180G, C180H, C1801,C180K, C180L, C180M, C180N, C180P, C180Q, C180R, C1805, C180T, C180V,C180W, and C180Y.
 88. The variant CBDA synthase or active fragmentthereof of claim 82, wherein one of the at least three amino acidmutations is at amino acid position
 414. 89. The variant CBDA synthaseor active fragment thereof of claim 88, wherein one of the at leastthree amino acid mutations at amino acid position 414 is selected fromthe group consisting of A414C, A414D, A414E, A414F, A414G, A414H, A414I,A414K, A414L, A414M, A414N, A414P, A414Q, A414R, A414S, A414T, A414V,A414W, and A414Y.
 90. The variant CBDA synthase or active fragmentthereof of claim 88, wherein one of the at least three amino acidmutations at amino acid position 414 is selected from the groupconsisting of: A414V and A414I.
 91. The variant CBDA synthase or activefragment thereof of claim 82, wherein one of the at least three aminoacid mutations is at amino acid position
 445. 92. The variant CBDAsynthase or active fragment thereof of claim 91, wherein one of the atleast three amino acid mutations at amino acid position 445 is selectedfrom the group consisting of I445A, I445C, I445D, I445E, I445F, I445G,I445H, I445K, I445L, I445M, I445N, I445P, I445Q, I445R, I445S, I445T,I445V, I445W, and I445Y.
 93. The variant CBDA synthase or activefragment thereof of claim 91, wherein one of the at least three aminoacid mutations at amino acid position 445 is I445M.
 94. The variant CBDAsynthase or active fragment thereof of claim 82, wherein the at leastthree amino acid mutations are H69Q, A414V, and I445M.
 95. A nucleicacid construct comprising a nucleic acid encoding the variantcannabinoid synthase or active fragment thereof of any one of claims1-94 operably linked to a promoter.
 96. A vector comprising the nucleicacid construct of claim
 95. 97. A microorganism comprising the nucleicacid construct of claim
 95. 98. The microorganism of claim 97, whereinthe microorganism is a yeast or a bacteria.
 99. The microorganism ofclaim 98, wherein the yeast is a Saccharomyces cerevisiae.
 100. Themicroorganism of claim 98, wherein the bacteria is an Escherichia coli.101. A plant comprising the nucleic acid construct of claim
 95. 102. Theplant of claim 101, wherein the plant is a vascular plant or anon-vascular plant.
 103. The plant of claim 102, wherein the vascularplant is a plant in the genus Cannabis.
 104. The plant of claim 103,wherein the plant is genus Cannabis is selected from the groupconsisting of: Cannabis satvia, Cannabis indica, and Cannabis ruderalis.105. The plant of claim 102, wherein the non-vascular plant is amicroalgae.
 106. A method of producing a cannabinoid, comprising: (i)contacting a cell with a nucleic acid encoding the variant cannabinoidsynthase of any one of claims 1-90, (ii) expressing the variantcannabinoid synthase, and (iii) isolating a cannabinoid produced by thecell.
 107. The method of claim 106, wherein the cell is a plant cell ora microorganism cell.
 108. The method of claim 106, further comprisingexpanding the cell to produce a plurality of expanded cells.
 109. Themethod of claim 107, wherein the expanding occurs in a bioreactor. 110.The method of claim 107, further comprising isolating and purifying thecannabinoid from the plurality of expanded cells.
 111. A method ofproducing a cannabinoid, comprising: contacting the variant cannabinoidsynthase of any one of claims 1-94 to a substrate of the variantcannabinoid synthase.
 112. The method of claim 111, wherein thecontacting occurs ex vivo.
 113. The method of claim 111, wherein thesubstrate is a naturally occurring substrate.
 114. The method of claim113, wherein the naturally occurring substrate is selected from thegroup consisting of cannabigerol (CBG), cannabigerolic acid (CBGA),cannbigerovarinic acid (GBGVA), and any homolog thereof
 115. The methodof claim 111, wherein the substrate is a non-naturally occurringsubstrate.
 116. The method of claim 115, wherein the non-naturallyoccurring substrate can comprise: a non-naturally occurring tailvariant, a prenyl donor, or a combination thereof.